Oxadiazole and phenol derivatives as antibacterial and/or herbicidal agents

ABSTRACT

Compounds of formula I 
                         
and II
 
                         
are described. In formulae I and II, X is —NH— or —S— and Y is —CH═ or —N═. In formula I, Z is C 1 -C 6  alkylene, C 2 -C 6  alkenylene, C 6 -C 10  arylene, or C 3 -C 6  cycloalkylene, or Z is absent. In formula I, R is hydrogen, halogen, —OH, —NO 2 , C 1 -C 6  alkyl, C 2 -C 6  alkenyl, C 6 -C 10 aryl, —O(C 1 -C 6  alkyl), or —O(C 6 -C 10  aryl), wherein each hydrogen atom in C 1 -C 6  alkyl, C 2 -C 6  alkenyl, C 6 -C 10  aryl, —O(C 1 -C 6  alkyl), and —O(C 6 -C 10  aryl) is independently optionally substituted by halogen, —OH, or —NO 2 . In formula II, R 1 , R 2 , R 3 , and R 4  are each independently hydrogen, halogen, —OH, —NO 2 , C 1 -C 6  alkyl, C 2 -C 6  alkenyl, or C 6 -C 10  aryl, wherein each hydrogen atom in C 1 -C 6  alkyl, C 2 -C 6  alkenyl, and C 6 -C 10  aryl is independently optionally substituted by halogen, —OH, or —NO 2 . Methods of inhibiting bacteria are also described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e)to U.S. Provisional Patent Application Nos. 62/476,260, filed Mar. 24,2017 and 62/518,947 filed Jun. 13, 2017. The disclosures set forth inthe reference applications are incorporated herein by reference in theirentireties.

The United States Government has rights in this invention pursuant toContract No. NIH R15 AI 113653 between the United States Government andthe Board of Trustees of Northern Illinois University.

BACKGROUND

Small molecule analogs of 2-(1H-indol-2-yl)-1,3,4-oxadiazole (also knownas FOL-7082) were synthesized and found to have improved antibacterialactivity by inhibiting the enzymes IspD and IspE in the MEP pathway.

Arguably one of the greatest medical developments is the discovery ofantibiotics. Antibiotics have not only helped increase the average humanlifespan and quality of life, but are also needed as defense againstbiological select agents and toxins (BSATs). Due to overuse andincorrect use of antibiotics, bacteria have developed resistance againstmost classes of antibiotics. Bacteria continue to mutate and developresistant genes. Discovering unique classes and mechanisms of action isa necessity for fighting back.

With the establishment of antibiotics came the generation ofantimicrobial resistance. Antibiotic resistance is a predominant threatwithin the health care system with estimated costs of greater than $20billion in the United States alone. Antibiotic resistance may evolvethrough natural selection with random mutations that are capable ofdeveloping a resistance gene that enables the bacteria to tolerateantibiotic treatment. With each antibiotic treatment there is anincrease in the resistant subpopulation, until the strain becomesprimarily drug-resistant.

Due to this resistance, the necessity of new antibiotics in which tofight these resistant strains of bacterial infections is dire. Despitethis fact, since the 1950s, new antibiotic approvals through the US Foodand Drug Administration (FDA) have drastically reduced each decade,which has culminated in a “discovery void” since the 1990s.

Approaches to new antibiotic development include analogs of knowncompounds or use of known classes that address known targets, newcompounds with new mechanisms of action within new classes that act onknown targets, and compounds within new classes acting on novel targets,

Antibacterial Targets

Each antibiotic class has a core structure related to its mode ofaction, with family members in the class possessing modifications aroundthe core. The different classes are commonly associated with theirtarget such as cell wall biosynthesis, protein synthesis, DNA and RNAsynthesis, folate synthesis, and disruption of cell membranes. Targetingand inhibiting these pathways halts a metabolic process that can lead tocell death.

1. Targeting Cell Wall Synthesis

One of the most common pathways for antibiotic development includesinhibiting the synthesis of bacterial cell walls and interfering withtheir structural integrity. This strategy has worked well, as mostbacteria possess a peptidoglycan cell wall while eukaryotic cells donot. Peptidoglycans are polymers of disaccharides which are cross-linkedby pentapeptide side chains. These polymers are maintained by a seriesof enzymes including transpeptidases, such as DD-transpeptidases, whichare targets for a number of antibiotics.

2. Inhibition of Protein Synthesis

The synthesis of proteins within the ribosomes of bacteria, is a majorantibiotics target. There are three phases in the translation processthat may be targeted: initiation, elongation and termination. Ribosomesare made up of two subunits, a larger 50s and a smaller 30s, whichassociate at the initiation step of translation.

Antibiotics targeting initiation at the ribosome level include compoundssuch as macrolides, lincosamides, aminoglycosides, tetracyclines,chloramphenicol, streptogramins, and oxazolidinones.

3. Inhibition of DNA or RNA Synthesis

Another pathway taken to an antibiotic is impeding DNA replication andRNA transcription.

4. Inhibiting Folate Synthesis

Folate is a compound necessary for cell division, DNA and RNA synthesisand methylation of DNA. Humans are unable to synthesize folates de novo;however, bacteria have the ability to biosynthetically create DNA basesfrom para-aminobenzoic acid (PABA) and pteridine.

5. Cell Membrane Disruption

Another pathway used for antibiotic development, is disruption ofcellular membranes. These membranes are composed of proteins andphospholipids, which allow selective permeability where bacteria take innutrients and dispel out waste. Disturbing this balance between nutrientand waste, through compromising the integrity of the cell membrane, is asuitable approach for antibiotics, which can lead to cell death.

Target Pathways in Bacteria

1. Isoprenoid Biosynthesis

Isoprenoids are a class of compounds that represent one of the mostdiverse group of metabolites in both structure and function. Isoprenoidsmake up more than 50,000 recognized molecules and are the oldest knownbiomolecules, with hopanoids being recovered from 2.5 billion year oldsediments. These molecules have a wide range of functions including theelectron transport chain quinones, membrane components such as prenyllipids and sterols found in archaebacteria and eubacteria, cellulartargeting agents seen in prenylation of proteins, carotenoids that serveas side chains in chlorophyll, hormones such as plant gibberellins,which promote growth, and as plant defense molecules, includingmonoterpenes, sesquiterpenes, and diterpenes. These compounds are allsynthetically derived from a 5-carbon subunit isopentenyl diphosphate(IPP) and dimethylallyl diphosphate (DMAPP) (FIG. 1). Examples of somecommon isoprenoids are shown in FIG. 2.

2. Mevalonic Acid (MVA) Pathway

Synthesis of IPP can occur via two different pathways. One pathway isthe cytoplasmic mevalonic acid (MVA) pathway (FIG. 3). The MVA pathway,which is present in humans, utilizes the precursor acetyl-CoA in whichthe first step involves the condensation of two acetyl-CoA moleculesthrough action of acetoacetyl-CoA thiolase to form acetoacetyl-CoA via aClaisen condensation reaction. This product is converted to3-hydroxy-3-methylglutaryl-CoA by HMG synthase. In two reduction steps,with NADPH as a reducing equivalent, 3-hydroxy-3-methylglutaryl-CoA isconverted to mevalonic acid (MVA) for which the pathway is named. Thesereductions are done by the enzyme 3-hydroxy-3-methylglutaryl-CoAreductase. Mevalonic acid is phosphorylated twice: once with MVA kinaseand a second time with phospho-MVA kinase (MVK and PMK respectively).This produces mevalonate-5-diphosphate. An ATP-dependent decarboxylationof MVA 5-diphosphate by MVA diphosphate decarboxylase (MPD) produces thedesired IPP subunit for isoprenoid synthesis.

3. 2-C-Methyl-D-Erythritol 4-Phosphate (MEP) Pathway

The second pathway that leads to IPP is utilized by many bacteria andsome higher plant life and is called the MEP pathway after one of theintermediates (2-C-Methyl-D-erythritol 4-phosphate). This pathway (FIG.4) was discovered and reported in 1999 using [C₁]glucose and [C]acetateisotopomers that seemed to be incorporated into hopanoids in ways notconducive to the traditional mevalonate pathway. In this pathway, theprecursor is D-glyceraldehyde-3-phosphate. In the first step, acondensation between the C1 aldehyde group ofD-glyceraldehyde-3-phosphate and (hydroxyethyl)thiamin (a derivative ofpyruvate) is catalyzed by 1-deoxy-D-xylulose-5-phosphate synthase (DXS).This reaction releases CO₂ and is irreversible. The product,1-deoxy-D-xylulose 5-phosphate undergoes an intramolecular rearrangementand reduction to produce 2-C-Methyl-D-erythritol 4-phosphate by actionfrom DXP reductoisomerase (IspC). MEP is converted to 4-(cytidine5′-diphospho)-2-C-methyl-D-erythritol (CDP-ME) by2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase (IspD) in aCTP-dependent reaction. Phosphorylation of a hydroxyl group at the C2position of CDP-ME by 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritolkinase (IspE) results in 2-phospho-4-(cytidine5′-diphospho)-2-C-methyl-D-erythritol (CDP-MEP). This is converted to2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcPP) by2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF). MEcPP isreduced by 4-hydroxy-3-methylbut-2-enyldiphosphate synthase (IspG) to4-hydroxy-3-methylbut-2-enyldiphosphate. Finally, this is converted toboth IPP and DMAPP by IspH, which can isomerize through action of IPPA-isomerase. DMAPP is an active molecule for condensation reactions withIPP.

Certain plants utilize both the MVA and MEP pathways and these are oftencompartmentalized, where MEP pathway reactions take place inchloroplasts and MVA pathway reactions in the cytosol, but some commonmetabolites can be exchanged between compartments. MEP pathway enzymesmay be targets of broad-spectrum antimicrobial and herbicidal agents orutilized by genetic modification of microorganisms to produce compoundsof medical and agricultural importance.

4. MEP Pathway as a Drug Target

The development of two separate pathways for isoprenoid biosynthesis isquite advantageous to the development of new compounds to combatparasitic and microbial infections. The enzymes present in the MEPpathway are potential targets for metabolic inhibition of variouspathogens, as there are no analogous enzymes present in humans. Theseenzymes in the MEP pathway are vital for many pathogens and theirfunction cannot be compensated by other enzymes present within the cell.In humans, inhibition of isoprenoid synthesis has become a subject ofresearch for treatment of hypercholesterolemia, osteoporosis, andcertain cancers. Humans utilize the MVA pathway, which uses differentenzymes than the MEP pathway. Isoprenoids are also required in bacteriaas essential components of electron transport and cell wallbiosynthesis. Gene knockout or enzyme inhibition with the antibioticfosmidomycin has shown that MEP pathway enzymes are necessary forcertain bacterial growth. As multi-drug resistance in bacteria becomes aproblem, it is quite beneficial to develop inhibitors of these enzymes.One of the most notorious pathogens that utilize the MEP pathway is themalaria parasite. Malaria is prevalent in many developing countries andis one of the most severe public health problems. About half of theworld's population (3.4 billion people) lives in high malaria riskareas. In 2012, there have been an estimated 207 million clinical casesof malaria infection, which resulted in around 627,000 deaths.

The malaria parasite has become resistant to typical antimalarial drugs,such as chloroquine, at a high rate and as such, new drugs are in urgentdemand.

a) Targeting IspD

The third enzyme in the MEP pathway, IspD, catalyzes the conversion ofMEP to CDP-ME by transferring the CMP from cytidine triphosphate (CTP)to the phosphate on the MEP, releasing pyrophosphate. Currently, thereare 35 crystal structures of IspD from 13 different species possessing31 different ligands deposited in the protein database (PDB). Mostnotably, a crystal structure of IspD bound to CTP and the reactionproduct CDP-ME, allows insight in the structure of the active site.

IspD is proposed to be active as a dimer with the subunits consisting ofa seven strand β-sheet with an α/β structure around it and an arm-likeloop that interacts with its neighboring monomer to form the observedhomodimer. Within the face of the dimer interface, there is a highlyconserved active site between species, specifically when comparing thesequences of Escherichia coli, Burkholderia thailandensis, Mycobacteriumtuberculosis, and Arabidopsis thaliana. Despite the presence of anydirect interactions with IspD residues, a divalent metal ion (most oftenMg²⁺) is necessary for the activity of the enzyme and is foundcoordinated to the α, β, and γ phosphates of CTP, likely stabilizing thereaction intermediate. Along with the Mg²⁺, residues Lys-27 and Lys-213aid in the coordination of the phosphates and are conserved acrossdifferent species. The base binding pocket is also conserved within theactive site involving hydrogen bonds being formed between the backboneamides from Ala-14 and Ala-15 with B. thailandensis having a cysteineinstead of an Ala-14. The carbonyl oxygen of Gly-82 also createshydrogen bonds and Asp-83 in E. coli, however, Asp-83 is not conservedbetween species. The hydroxyl groups of the ribose ring coordinate withPro-13, Gly-16 and Ala-107 which are completely conserved between E.coli, B. thailandensis and M. tuberculosis, Pro-13 and Ala-107 areVal-13 and Ser-107 respectively for A. thaliana.

4-Diphosphocytidyl-2C-methyl-D-erythritol synthase, or IspD, is thethird enzyme in the MEP pathway that is utilized to catalyze theformation of 4-diphosphocytidyl-2C-methyl-D-erythritol by transfer of adiphosphocytidyl unit from CTP to 2-C-methylerythritol 4-phosphate, orMEP (FIG. 7).

b) IspD (Mycobacterium paratuburculosis)

The structure of IspD is a homodimer with a globular domain consistingof α/β folds. IspD contains a highly conserved active site in eachmonomer among various species of bacteria and plants. Most residues ofthe active site belong to the main globular domain, but some are locatedin the β-arm. Each active site (2 active sites for the dimer) is foundat the dimer interface, making it relatively exposed to solvent and as aresult, competitive inhibitors to the natural substrate are difficult tomake as the active site is found in a polar environment. (FIG. 8)

The catalytic activity of IspD requires the presence of divalentcations, of which Mg²⁺ is optimal for many bacterial species such asMycobacterium tuberculosis and Escherichia coli. Mg²⁺ coordinates withthe enzyme first. After Mg²⁺, CTP binds, followed by MEP. The proposedrole of the active site amino acids is shown in FIG. 9.

IspD, the third enzyme in the non-mevalonate pathway, catalyzes thecreation of if 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) which isthe substrate for IspE. This in turn creates CDP-MEP with ATP.Inhibiting these enzymes would disrupt the formation of isoprenoidsneeded for biological function.

c) Targeting IspE

The fourth enzyme in the MEP pathway (IspE) catalyzes the adenosinetriphosphate (ATP) dependent phosphorylation of CDP-ME to CDP-MEP. Thiskinase is also an attractive target for inhibition to disrupt thebiosynthesis of isoprenoids. To date, there are 17 crystal structures ofsix different species of IspE deposited in the PDB with 17 differentligands.

IspE is commonly reported as a homodimer, with each monomer comprisingof 10 α-helices and 12 β-sheets. Monomers dimerize with salt bridgesformed between Arg-21 of one monomer and Asp-80 on the other monomer,along with hydrogen bonds formed between the amide of Gly-87 and thecarbonyl of Ala-22. The dimer results in a solvent-filled channel withactive sites at each end of the channel. Each subunit has two domains;one domain provides a binding site for ATP, the second provides abinding site for the substrate CDP-ME.

Active sites are highly conserved between species, with 100% sequencehomology in the CDP-ME binding site and a 75% sequence homology in theATP binding site between E. coli and B. thailandensis IspE. The ATPbinding subunit comprises of a purine binding pocket which involveshydrogen bonding to Asn-65 and Asn-110 on either side of the adeninebase along with Leu-66 and Lys-96. The ribose ring and phosphates aresolvent exposed with the phosphate portion forming hydrogen bonds withthe amides in a glycine rich loop (Gly-103, Gly-105 and Gly-107) andSer-108.

The second subunit, in which CDP-ME binds to, comprises of two aromaticside chains (Phe-185 and Tyr-25) in which the cytosine portion of theCDP-ME will coordinate in between along with hydrogen bonds with His-26.The ribose ring and phosphates are again solvent exposed with the α andβ-phosphates of the CDP-ME directed to the γ-phosphate of the ATP. Theoxygen linker between the α and β-phosphates of the CDP-ME iscoordinated by a hydrogen bond with Ala-140. Hydrogen bonding with thehydroxyl on the methylerythritol involves Asp-141, Lys-10 and Asn-12.

d) ISPE (Mycobacterium abscessus)

4-diphosphocytidyl-2C-methyl-D-erythritol kinase, or IspE, is the fourthenzyme in the MEP pathway. It is a kinase that catalyzes the transfer ofthe γ-phosphoryl group of ATP to 4-diphosphocytidyl-2-C-methylerythritolvia the 2-hydroxyl group (FIG. 10).

IspE belongs to the galactose/homoserine/mevalonate/phophomevalonatekinase superfamily (GHMP). IspE is a monomer in solution and contains atwo-domain fold which contains an ATP and substrate binding domain andthe active site is found in a deep pocket between the two domains, whichis stabilized by water-mediated interactions after the substrate binds.

Fragment Hit Identification of FOL7082

An initial screening of a small molecule fragment library by the SeattleStructural Genomics Center for Infectious Disease was performed by Dr.Darren Begley using a saturation transfer difference NMR technique. Thismethod utilizes the nuclear Overhauser effect (NOE) and it relies on thefact that a weak-binding ligand will exchange between a bound and freeligand state. A ¹H-NMR experiment is performed on a free ligand insolution with the protein of interest without a saturation pulse(Off-resonance). A subsequent ¹H-NMR experiment is done in which thesolution is irradiated with a saturation pulse that is selective to theprotein, typically at 0 ppm to −1 ppm (On-resonance). By irradiating thesample at a selective frequency, the protein or receptor will becomesaturated and the signal intensity will diminish. Saturation istransferred through the protein/receptor to the fragment if the fragmentis within 5 Å of the protein in a binding event. The saturation-transferdifference is a measure of the difference between signal intensities ofthe ligand in the off-resonance state, I₀, and the signal intensitiesfollowing the saturation pulse, I_(SAT). If the fragment bound tightly,the I_(SAT) will be close to zero and the difference between I₀ andI_(SAT) is near I₀. If a part of the ligand did not bind to the proteinand there was little saturation transferred, I_(SAT) will approach I₀,and I₀-I_(SAT) will approach 0. By subtracting the on-resonance spectrumfrom the off-resonance spectrum, a saturation transfer difference (STD)spectrum is generated.

The peak intensities for the ligand are directly proportional to thedegree of binding to the protein/receptor. By using this data, a seriesof small molecule fragments were identified to bind to various enzymesin the MEP pathway.

In total, 1020 fragments were screened from the “Fragments of Life”compound library against the IspD from Mycobacterium paratuberculosis,IspE from Mycobacterium abscessus, and IspF from Burkholderiapseudomallei. Of the 1020 compounds, 102 were found to bind to MpIspD,176 were found to bind to MaIspE, and 81 were found to bind to BpIspF.Of these, 37 bound both to MpIspD and MaIspE. (see Table 6, and FIG. 6)

One of the shortcomings of utilizing STD-NMR is that the exact bindingmode of the fragments cannot be determined without further experimentsand the strength of binding is unknown without further assay data. Ofthe 37 dual binding fragments, FOL7082 was further explored to increasebinding efficiency using a Topliss tree approach (described herein).

SUMMARY

New insights are provided into the biophysical properties of IspD andIspE enzymes and methods to evaluate compounds with potential inhibitoryactivity. A high throughput thermal shift assay was designed to assessinhibitors for further studies. Enzymatic assays were developed tofurther study compounds for both IspD and IspE. Inhibitors of Ec IspDwere discovered to be in high micromolar range from the MicroSourceSpectrum collection which are more potent than low millimolar range.IspE from both E. coli and B. thailandensis were found to have severalinhibitors from the oxadiazole series in the low micromolar range. Noinhibitors against B. thailandensis IspE have been reported, making itan attractive target.

Analogs of 2-(1H-indol-2-yl)-1,3,4-oxadiazole (also known as FOL-7082)were synthesized and found to inhibit the EclspD (Escherichia coli) andBtIspE (Burkholderia thailondensis) enzymes. Antibacterial activity ofthe analogs against 9 strains of bacteria was determined Some analogshave activity against Pseudomonas aeruginosa and other bacteria at about150 micrograms/milliters (Kirby-Bauer assay).

From the variety of assays utilized to evaluate the compound library ofanalogs, the general trend observed is that a phenyl ring substitutionon the 1,3,4-oxadiazole was beneficial, and when the phenyl wassubstituted at the 4 position, activity was strongly enhanced for IspDand IspE. Depending on the species of IspD targeted, an electrondonating or electron withdrawing substituent enhanced activity greatly.Utilizing the 4-substituted phenyl compounds that had greatest activityin the indole series, a benzothiazole series was synthesized whichresulted in a significant loss in activity, likely due to loss of thehydrogen bond donating indole N—H or the electron withdrawing effects ofthe heterocycle. When a benzothiazole series was made, activity wasgreatly enhanced as in addition to the hydrogen bond donor, a hydrogenbond acceptor was also added compared to the indole parent compound.

Compound HGN-340, not an oxadiazole, was also found to haveantibacterial activity, possibly through inhibition of the IspD enzyme.HGN-340 showed significant inhibition of AtIspD, and analogs of thiscompound are predicted to be beneficial. Because this compound was madeto be an analog by changing the brominated pyrolle to an indole ring ofa reported allosteric inhibitor of AtIspD, an x-ray crystal structure isavailable with the pentabromopseudilin bound. A logical next step is touse computational methods to dock HGN-340, as well as analogs of HGN-340and compare docking scores as a means of designing new analogs. Thesecan then be synthesized and tested.

HGN-333 did show activity against IspD, IspE, and IspF in antibacterialassays and showed significant antibacterial activity.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the office upon request and paymentof the necessary fee.

FIG. 1A-1B: Structures of Isopentenyl Diphosphate and DimethylallylDiphosphate-FIG. 1A Isopentenyl diphosphate and FIG. 1B dimethyallyldiphosphate, two products of the 2-C-methyl-D-erythritol 4-phosphate(MEP) pathway and the mevalonic acid (MVA) pathway.

FIG. 2A-2D. Examples of Isoprenoid Compounds. Examples of IsoprenoidCompounds—Isoprenoids represent a large class of compounds derived froma common subunit. Some examples of common isoprenoids are FIG. 2Acholesterol (important component of cell membranes, FIG. 2B ubiquinone(involved in electron transport chain in mitochondria), FIG. 2Cgibberellins (plant hormones that are involved in growth anddevelopment), and FIG. 2D beta-carotene (a precursor to vitamin A).

FIG. 3. MVA Pathway—The MVA pathway is utilized by mammals and otherhigher eukaryotes in synthesis of isoprenoid subunits IPP and DMAPP.

FIG. 4. MEP Pathway, an alternate pathway to the MVA pathway that isfound in many bacteria, plants, and parasites such as malaria.

FIG. 5. Schematic of Saturation Transfer Difference NMR—Schematic ofSaturation Transfer Difference NMR—A technique used to identify smallmolecules that bind to a protein through subtraction of the on-resonancespectra from the off-resonance spectra. The difference shows parts ofthe molecule that are involved in binding. Reprinted with permissionfrom Viegas, A., et al. Saturation-Transfer Difference (STD) NMR: ASimple and Fast Method for Ligand Screening and Characterization ofProtein Binding. Journal of Chemical Education 2011, 88, 990-994.Copyright 2011 American Chemical Society.

FIG. 6A-6D. STD-NMR Fragment Screening Results (SSGCID). FIG. 6A:FOL7185; FIG. 6B: FOL 7082; FIG. 6C: FOL 7380; FIG. 6D: is a schematicof a number of binding sites of IspD, E, F in T. gondii (control).

FIG. 7. IspD Reaction—IspD catalyzed transfer of CMP to MEP to form4-diphosphocytidyl-2-C-methylerythritol.

FIG. 8. X-Ray Crystal Structure of Mycobacterium tuberculosis IspDHomodimer with Mg²⁺ (orange) and CTP (red) Bound; PDBID 2XWN.

FIG. 9. Proposed Role of Amino Acids in Binding ofMEP-cytidyltransferase Intermediate. Reprinted with permission fromRichard, S. B., et al. Kinetic Analysis of Escherichia coli2-C-Methyl-d-erythritol-4-phosphate Cytidyltransferase, Wild Type andMutants, Reveals Roles of Active Site Amino Acids. Biochemistry 2004,43, 12189-12197. Copyright 2004 American Chemical Society.”

FIG. 10. IspE Reaction—IspE catalyzed transfer of phosphoryl group fromATP to 4-diphosphocytidyl-2-C-methylerythritol to form4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate.

FIG. 11. X-Ray Crystal Structure of Mycobacterium abscessus IspE withATP (red) and CMP (blue) Bound to Active Site; PDBID 4DXL.

FIG. 12. Topliss Tree for Phenyl Substitutions—H designates anunsubstituted compound depending on activity of 4-Cl, different routescan be taken to obtain more potent compounds.

FIG. 13. Topliss Tree for Aliphatic Substitutions—H designates anunsubstituted compound and methyl is the first substitution followed byisopropyl. Depending on how moving from methyl to isopropyl affected theactivity, the appropriate route of synthesis would be taken.

FIG. 14A-14B. Topliss Tree Sets for FOL7082. Compound set was designedby following the Topliss Tree scheme beginning with FOL7082,unsubstituted at the 5 position on the 1,3,4-oxadiazole ring.

FIG. 15A-15C. Pentabromopseudilin—FIG. 15A, Pentabromopseudilinstructure and FIG. 15B, IspD bound configuration of pentabromopseudilinin X-ray crystal structure with Arabidopsis thaliana IspD. Due to acidicnature of the tribromo substituted pyrrole and dibromo substitutedphenol, these compounds were able to coordinate to Mg²⁺. Crystalstructure reprinted with permissions from Wiley Online Library. FIG.15C, Alignment of Arabidopsis thaliana IspD (PDBID 4NAK, light blue)with pentabromopseudlin(red) x-ray crystal structure and Arabidopsisthaliana IspD (PDBID 1W77, green) with cytidine-5′-monophosphate (darkblue). This shows the proximity of the allosteric binding site ofpentabromopseudilin to the active site in a monomer of IspD.

FIG. 16. Preparation of 1H-indole-2-carbohydrazide

FIG. 17. Preparation of 2-(1H-indol-2-yl)-1,3,4-oxadiazole (HGN-207)

FIG. 18. Preparation of 2-(1H-indol-2-yl)-5-phenyl-1,3,4-oxadiazole(HGN-208)

FIG. 19. Preparation of 2-(1H-indol-2-yl)-5-methyl-1,3,4-oxadiazole(HGN-209)

FIG. 20. Preparation of2-(1H-indol-2-yl)-5-(trifluoromethyl)-1,3,4-oxadiazole (HGN-211)

FIG. 21. Preparation of2-(1H-indol-2-yl)-5-(methoxymethyl)-1,3,4-oxadiazole (HGN-212)

FIG. 22. Preparation of2-(4-chlorophenyl)-5-(1H-indol-2-yl)-1,3,4-oxadiazole (HGN-213)

FIG. 23. Preparation of2-(1H-indol-2-yl)-5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazole(HGN-214)

FIG. 24. Preparation of 2-(1H-indol-2-yl)-5-phenethyl-1,3,4-oxadiazole(HGN-215)

FIG. 25. Preparation of 2-benzyl-5-(1H-indol-2-yl)-1,3,4-oxadiazole(HGN-221)

FIG. 26. Preparation of2-(1H-indol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazole (HGN-223)

FIG. 27. Preparation of2-(tert-butyl)-5-(1H-indol-2-yl)-1,3,4-oxadiazole (HGN-0239)

FIG. 28. Preparation of 2-(1H-indol-2-yl)-5-isopropyl-1,3,4-oxadiazole(HGN-240)

FIG. 29. Preparation of2-(3-chlorophenyl)-5-(1H-indol-2-yl)-1,3,4-oxadiazole (HGN-241)

FIG. 30. Preparation of 2-(1H-indol-2-yl)-5-(p-tolyl)-1,3,4-oxadiazole(HGN-242)

FIG. 31. Preparation of3-(5-(1H-indol-2-yl)-1,3,4-oxadiazol-2-yl)-N,N-dimethylaniline (HGN-249)

FIG. 32. Preparation of 2-ethyl-5-(1H-indol-2-yl)-1,3,4-oxadiazole(HGN-250)

FIG. 33. Preparation of 2-cyclobutyl-5-(1H-indol-2-yl)-1,3,4-oxadiazole(HGN-251)

FIG. 34. Preparation of 2-cyclopentyl-5-(1H-indol-2-yl)-1,3,4-oxadiazole(HGN-252)

FIG. 35. Preparation of2-(3,4-dichlorophenyl)-5-(1H-indol-2-yl)-1,3,4-oxadiazole (HGN-253)

FIG. 36. Preparation of 2-cyclohexyl-5-(1H-indol-2-yl)-1,3,4-oxadiazole(HGN-254)

FIG. 37. FIG. 36: Preparation of2-([1,1′-biphenyl]-3-yl)-5-(1H-indol-2-yl)-1,3,4-oxadiazole (HGN-287)

FIG. 38. Preparation of2-([1,1′-biphenyl]-4-yl)-5-(1H-indol-2-yl)-1,3,4-oxadiazole (HGN-288)

FIG. 39. Preparation of benzo[d]thiazole-2-carbohydrazide.

FIG. 40. Preparation of2-(benzo[d]thiazol-2-yl)-5-((2,4-dichlorophenoxy)methyl)-1,3,4-oxadiazole(HGN-333)

FIG. 41. Preparation of2-(benzo[d]thiazol-2-yl)-5-(3,5-dinitrophenyl)-1,3,4-oxadiazole(HGN-334)

FIG. 42. Preparation of(E)-2-(benzo[d]thiazol-2-yl)-5-styryl-1,3,4-oxadiazole (HGN-335)

FIG. 43. Preparation of2-(benzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazole (HGN-336)

FIG. 44. Preparation of2-(benzo[d]thiazol-2-yl)-5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazole(HGN-337)

FIG. 45. Preparation of2-(benzo[d]thiazol-2-yl)-5-(p-tolyl)-1,3,4-oxadiazole (HGN-338)

FIG. 46. Preparation of2-(benzo[d]thiazol-2-yl)-5-(4-chlorophenyl)-1,3,4-oxadiazole (HGN-339)

FIG. 47. Preparation of 2,4-dibromo-6-(1H-indol-2-yl)phenol (HGN-340)

FIG. 48. Preparation of2-(1H-benzo[d]15midazole-2-yl)-5-(4-chlorophenyl)-1,3,4-oxadiazole(HGN-421)

FIG. 49. Preparation of2-(1H-benzo[d]imidazol-2-yl)-5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazole(HGN-422)

FIG. 50A-50B. Thermal shift data for FIG. 50A, MpIspD and FIG. 50BEclspD. Values are ΔT_(m) given in ° C. for the unfolding event.

FIG. 51A-51B. Results from Washington University School of Medicine IspDAssay (Dr. Audrey Odom)—Results are shown as a inhibition curve wherethe x-axis represents compound concentration and the y-axis represents %activity of IspD enzyme. Data is also represented in a bar graphrepresenting reaction rate of IspD at FIG. 51A, 12.5 and FIG. 51B, 100μM compared to IspD without inhibitor.

FIG. 52. Reaction Scheme for University of Hamburg Assay AgainstIspD-Reaction scheme involves coupled reactions that allow consumptionof NADH to be monitored at 340 nm to assess reaction rate.

FIG. 53. Reaction Scheme for University of Hamburg Assay AgainstIspE-Reaction scheme involves coupled reactions that allowed consumptionof NADH to be monitored at 340 nm to assess reaction rate.

FIG. 54. Reaction Scheme for University of Hamburg Assay AgainstIspF-Reaction scheme involves coupled reactions that allowed consumptionof NADH to be monitored at 340 nm to assess reaction rate.

FIGS. 55A-55F. Results of an IspD assay on E. coli: FIG. 55A, EclspDHGN-211; FIG. 55B, EclspD HGN-212; FIG. 55C, EclspD HGN-213; FIG. 55D,EclspD HGN-214; FIG. 55E, EclspD HGN-215; FIG. 55F, EclspD HGN-221.

FIGS. 56A-56F. Results of an IspD assay on P. falciparum: EclspDHGN-211; FIG. 56B, EclspD HGN-212; FIG. 56C, EclspD HGN-213; FIG. 56D,EclspD HGN-214; FIG. 56E, EclspD HGN-215; FIG. 56F, EclspD HGN-221.

DETAILED DESCRIPTION

The 2-C-methyl-D-erythritol-4-phosphate pathway, or MEP pathway, hasrecently gained attention as being a prime target for inhibition of manypathogenic species due to the lack of analogous enzymes in humans andmany higher eukaryotes that use the mevalonic acid pathway. Either theMEP pathway or mevalonic acid pathway are utilized by all livingorganisms to generate the molecules isopentenyl diphosphate, IPP, anddimethyallyl diphosphate, DMAPP, which in turn can be used to create awide variety of necessary cellular components called isoprenoids, suchas cholesterol, some vitamins, pigments. The MEP pathway involves sevendifferent enzymes, which are unique in the cellular machinery. Each is apotential target for inhibition by small molecules that could prove tobe fatal to the organism.

In an NMR based screening of the Fragments of Life library, the fragmentFOL7082 was one of thirty-seven compounds that bound to both IspD ofMycobacterium paratuburculosis and IspE of Mycobacterium abscessus. Thefragment hit FOL7082 was optimized using a Topliss Tree approach togenerate a small library of analogs that were tested in a variety ofbiological assays. Synthesis of this library was performed using areaction scheme that allowed for a high number of analogs to be madefrom the same starting material with only variation of the carboxylicacid used. Further changes to FOL7082 were made by incorporatingdifferent heterocycles in place of the indole, such as benzothiazole andbenzimidazole, to test if there was an increased activity againstenzymatic targets IspD and IspE.

Overall, analogs of the original fragment hit, FOL7082 were synthesizedthat resulted in a greater than 20-fold increase in activity againstenzyme target IspD. Analogs showed some antibacterial activity as wellas herbicidal activity through inhibition of germination. The projectwas successful in taking a fragment hit with no measurable activity to acompound with 50 μM activity against IspD.

A goal was to discover novel hit compounds targeting IspD or IspE for E.coli and B. thailandensis. Currently, there are very few knowninhibitors of either IspD or IspE with most possessing IC₅₀ values inthe millimolar range.

The biophysical properties of IspD and IspE were investigated to aiddiscovery of novel inhibitors. Saturation transfer difference nuclearmagnetic resonance (STD-NMR) fragment screening identified severalfragments that bound to both IspD from Mycobacterium paratuberculosisand IspE from Mycobacterium abscessus. Analogs of these fragments aimedto target IspD and IspE. Thermal shift screening was used as a filter toidentify a subset of compounds that bind to the IspD and IspE enzymes.High throughput thermal shift screening of the MicroSource Spectrumlibrary against EclspD revealed several potential hits, which weresubsequently investigated using a plate-based enzyme inhibition assay.

Results from the enzyme inhibition assays revealed a small set ofcompounds capable of inhibiting IspD activity. Follow-up studies wereperformed using isothermal titration calorimetry and enzymaticinhibition assays. Results from these studies have confirmed compoundswith high micromolar inhibition targeting IspD from Escherichia coli.The IspE enzyme from both Escherichia coli and Burkholderiathailandensis were characterized with biophysical methods. Additionally,a plate-based enzyme inhibition assay was optimized to evaluatepotential IspE inhibitors, which led to the discovery of several novelinhibitors (IC₅₀ values in the micromolar range). The identifiedcompounds appear promising for future development of even more potentIspD and IspE inhibitors.

The high throughput thermal stability assays of EclspD were used toassess binding potencies of over 2,700 biologically active andstructurally diverse compounds that are either known drugs, experimentalbioactive agents, or pure natural products. The compounds that had athermal shift, indicating some interaction, were evaluated further withan enzymatic assay to determine activity against EclspD. Finally, thesame techniques were used to study IspE from both E. coli and B.thailandensis with the focus of future inhibitor design.

To expand the development of novel chemical matter, beyond what wasidentified using NMR screening, compounds were evaluated for bindingactivity against IspD using a thermal shift assay a library ofcompounds. The MicroSource Spectrum collection contains about 2,500compounds of known drugs and natural products. Initial thermal shiftresults showed stability shifts for 48 compounds, both positive andnegative shifts with a range of +3.53° C. (thimerosal) to −13.53° C.(cadmium acetate). Compounds were evaluated further using the enzymaticactivity assay, which revealed a few compounds with high micromolaractivity: isoliquiritigenin (IC₅₀=279±12 μM), thimerosal (IC₅₀=381±12μM), and carnosic acid (IC₅₀=794±100 μM). While these activities are notvery potent, they are more potent than the known E. coli inhibitorswhich could be used for future development for more potent inhibitors.

Initial thermal stability studies provided insight on the melting pointsof E. coli and B. thailandensis IspD. A thermal shift assay is anefficient and cost effective way of evaluating a series of compounds asan initial filter to determine which were evaluated further forenzymatic activity. Current inhibitors of Ec IspD mimic the substrate,such as fosmidomycin. NMR fragment screening of the Fragments of Lifelibrary against IspD from Mycobacterium paratuberculosis and IspE fromMycobacterium abscessus showed 102 compounds bound IspD and 176compounds bound IspE. Of these known binders, 37 compounds bound to bothIspD and IspE. One of those fragments that bound to both IspD and IspEis the base of the oxadiazole series which was studied with the thermalshift assay against Ec IspD. A significant shift in the thermalstability was not shown. Another class of fragment hits for both enzymeswas the pyrazolopyrimidines, which again showed no significant thermalstability shifts. Most of these compounds were evaluated further usingan enzymatic activity assay. These compounds showed weak to no activityagainst EclspD. Isothermal titration calorimetry revealed that thereactant CTP binds with weak affinity to EcIspD, which could indicatewhy compounds have such a weak affinity for IspD.

Similar to IspD, IspE was analyzed and characterized using the sametechniques against the pyrazolopyrimidine series and oxadiazole seriesof compounds. Thermal shift results against B. thailandensis ranged from+5.5° C. (pyrazolopyrimidine with furan functional group) to −4.5° C.(pyrazolopyrimidine with dichlorobenzene). An enzymatic assay wasdeveloped and optimized to evaluate the IspE against E. coli and B.thailandensis further revealing several compounds in the low micromolarrange. The pyrazolopyrimidine series only showed a few compounds in thehigh micromolar range, however the oxadiazole series proved morefruitful for a range of activity from high to low micromolar. The mostpotent compound for both species was HGN-333, an oxadiazole with a2,4-chloro benzene with an IC₅₀ for E. coli and B. thailandensis of3.0±0.3 and 28.0±6.5 04, respectively. This compound is an idealcandidate for further development of more active compounds.

Example A—Biochemical and Structural Characterization of Ispd andEvaluation of Inhibitors

There is a great need for discovering new inhibitors of this enzyme dueto the lack of potent inhibitors, specifically against E. coli and B.thailandensis. Binding activity along with enzymatic activity ofcompounds can be determined with a variety of methods but each haslimitations, consequently, the use of multiple techniques often provesmost useful. Thermal stability assays, isothermal titration calorimetry,and enzymatic activity assays were used to assess potential smallmolecule inhibitors. Thermal stability assays give insight of howcompounds may influence protein stability due to ligand interactions.However, thermal stability assays do not provide information on thelocation of the binding site, nor does binding equate to inhibition ofenzymatic activity. Isothermal titration calorimetry (ITC) can providethe thermodynamic parameters of the ligand-enzyme interaction, includingK, ΔG°, ΔH°, ΔS°, and the binding stoichiometry. However, as with mosttechniques, the ability to obtain weak binding affinity dissociationconstants (K_(D) values in mM range) with ITC it is limited by the smallmolecule solubility, as well as the amount of sample required pertitration. Finally, enzymatic inhibition activity assays reveal the keyproperty, the ability of a compound to inhibit the enzyme. Such assayscan also be run in a high throughput fashion, however, limitations arisedue to the expensive substrates and timely experiments. A series ofcompounds against IspD from E. coli and B. thailandensis were based offfragment hits from a STD-NMR screening performed by Dr. Darren Begley(SSGCID).

This technique studies the interactions between proteins and ligandswith the Nuclear Overhauser Effect (NOE) by selectively saturatingprotons within the protein without the ligand which creates a referencespectrum. If a ligand binds to the protein, spin diffusions will causethe saturation to disperse to the ligand, which would reduce the signal.Subtracting the resulting spectrum from the reference spectrum providesa spectrum for the binding ligands showing which portion of the ligandare interacting with the protein.

Results

Circular dichroism thermal stability studies of IspD for both E. coliand B. thailandensis have shown IspD has melting temperatures suitablefor thermal shift assays. Thermal shift assays were performed onpotential inhibitors from the pyrazolopyrimidine and the oxadiazoleseries which were designed off the fragment hits from the STD-NMRscreening. The thermal shift studies did not show any significantstabilization or destabilization caused by the compounds on IspD. Thereare several reasons why there was no observed stabilization changes tothe IspD enzyme. These thermal shift assays were performed against adifferent species of IspD compared to the binding studies of the STD-NMRscreening which was performed against M. tuberculosis. Anotherpossibility is that the thermal shift assay compound concentration was40 μM which could be too low of a concentration to show anystabilization or destabilization to the enzyme considering knowninhibitors bind to the active site in the millimolar range. An enzymaticassay was optimized for Ec IspD to evaluate several of the compounds todetermine if any have any inhibition activity, however there was nosignificant activity shown. ITC results of Bt IspD with CTP revealedweak binding to the cofactor. This weak binding could indicate why theknown inhibitors have such a weak IC₅₀ values against IspD.

Example B: Discovery of Small Molecule Enzyme Inhibitors of E. Coli IspdVia High-Throughput Screening

Within drug discovery, there are two main strategies for discovering hitcompounds: knowledge-based design and random screening. Knowledge-baseddesign is based on information of the target, which could be a knowninhibitor or substrate that can be mimicked, or perhaps a crystalstructure of a compound/protein complex. Conversely, random screeningdoes not require prior knowledge of the target, and requires screeninglibraries of compounds with a variety of structures and bioactivity.

The MicroSource Spectrum library was screened using the thermalstability assay to evaluate 2,560 compounds which consists of 1040 drugsthat have made clinical trials stage in the US, 240 international drugsthat were marketed in Europe and Asia, and the remaining compounds areeither natural products or derivatives of the other. The compounds thatshowed a shift in the stability of IspD were evaluated further using theEnzChek® enzymatic activity assay.

Thermal Shift Results

The library based screening technique in conjunction with the thermalshift assay was implemented to evaluate compounds from Spectrumcollection. Out of the 2,560 compounds there were 48 hit compounds thatdisplayed a change in the observed melting temperature, ranging from+3.53° C. (thimerosal) to −13.53° C. (cadmium acetate). Thimerosal(+3.53° C.) and Zinc Pyrathione (−7.72° C.) were evaluated further basedon the clarity of the unfolding profile. Specifically, each compound wasbroken down into core structures to determine which portion of thecompound affected the thermal stability. To study thimerosal,thiosalicylic acid and ethyl mercuric chloride were evaluated. Theresults of the assay revealed that the ethyl mercuric chloride portionof thimerosal affected the stability of the enzyme. Zinc pyrathione wassimplified to 2-mercaptopyridine-N-oxide to determine if the zinc wasthe driving force for the destabilization of Ec IspD. Thermal shiftresults revealed that 2-mercaptopyradine-N-oxide did not have an effecton the thermal stability. Therefore, similar to the thimerosal, itappears that the metal is the prime factor on the stability of theenzyme.

EnzChek® Assay Results

The 48 compounds, which altered the stability of Ec IspD, were evaluatedfurther with an enzymatic assay to determine potential inhibitoryactivity. Dose response curves found three compounds with IC₅₀ values inthe high micromolar range. Thimerosal, which was used as a preservativein vaccines, showed an IC₅₀ of 381±12 μM. Isoliquiritigenin, a naturalchalcone, has an IC₅₀ of 279±12 μM. Carnosic acid, a preservative infood, showed an IC₅₀ of 794±100 μM. The EnzChek® assay has a phosphatesensitivity of 2-150 μM. To ensure there is no interference caused byassay components, three standards were performed on each plate: bufferonly, without CTP and without MEP. These standards confirmed that therewas no phosphate contamination.

DISCUSSION

The screening of compound libraries is an invaluable tool for hitdiscovery. This technique allows screening against a wide variety ofchemical structures, beyond the knowledge-based compounds developed. TheMicroSource Spectrum Collection was evaluated against Ec IspD using thethermal stability assay, and 48 changes in stabilization were observed.The range of thermal shifts were from +3.53 (thimerosal) to −13.53(cadmium acetate). Thimerosal and zinc pyrathione were selected foradditional studies due to their unfolding profiles. Each of these twocompounds were dissected, which revealed that the metals in thecompounds were the driving force for the thermal stability change.

An enzymatic assay was performed against the 48 compounds that showed astability change to determine if any had any activity against Ec IspD.Three compounds displayed inhibition in the high micromolar range:thimerosal (381±12 μM), isoliquiritigenin (279±12 μM) and carnosic acid(794±100 μM). Thimerosal, which shows modest activity against the EcIspD, is a well-known organomercurial preservative that has been subjectto much controversy of its toxicity, components its use has been greatlyreduced over the years and some countries have banned its use entirely.This compound may not be ideal for future work. Carnosic acid, a threering abietane found in rosemary, has shown to have antimicrobialproperties and is commonly found in oral care products such as mouthwashand toothpaste. This compound, which also has modest activity, is not anideal candidate for further investigation due to the catechol moietywhich tends to create prolific binding properties, reducing specificity.Isoliquiritigenin, a chalcone found in licorice, has shown to have awide array of properties from anti-cancer to anti-inflammatoryactivities. This compound would be the most ideal candidate, out of thethree examined, to investigate further as possible lead compound.

Example C: Biochemical and Structural Characterization of Ispe andEvaluation of Inhibitors

Currently, IspE has several known inhibitors in the low to midmicromolar range against E. coli, however there are no known inhibitorsagainst B. thailandensis, making it an attractive target for novelinhibitor discovery.

The two chemical series that were identified from the NMR fragmentscreening, the pyrazolopyrimidines and oxadiazoles, were evaluatedfurther using the thermal shift assay. An enzymatic assay was developedand optimized to study these compounds for activity against both E. coliand B. thailandensis. ITC will determine dissociation constants ofpromising compounds.

DISCUSSION

The two series of compounds: pyrazolopyrimidines and oxadiazoles, werestudied against IspE using a series of assays to determine binding andactivity. Thermal shift assays were performed to determine a potentialchange of stability to IspE from B. thailandensis. All but one of thepyrazolopyrimidines showed little to no significant shifts, onlyHGN-0377 (furan) having a +4.0° C. shift. Oxadiazoles showed to have alarger range of stability shifts with the largest shift of +6.5° C.which was HGN-0813 (standard). These compounds were evaluated withenzymatic assays to determine activity against both E. coli and B.thailandensis. Similar to the thermal assay, the pyrazolopyrimidines hadvery little activity against both species from the enzymatic assay. Theoxadiazoles had greater activity with HGN-333 (2,4-dichloro) having anIC₅₀ of 3.0 μM against Ec IspE and 28.0 μM against Bt IspE. To ensurethat this compound was not interfering with the luciferase, the reactionwas run without the IspE which showed no change in the signal comparedto the control containing all reactants minus the enzyme and a compound.ITC experiments also verify binding of HGN-333 showing a K_(D) of2.2E+06±1.5E+06 and a −TΔS of −17.8 kcal/mol which suggests that thereaction is entropically driven which agrees with predictions oflipophilic pockets.

Results of Biological Assay Evaluation of Topliss Tree Compound Set

1. Thermal Shift Assay

Thermal shift assays are often used to determine the relative bindingefficiency of low molecular weight compounds to an enzymatic targetthrough measurement of changes in enzyme melting point temperatureswithout the ligand and with the ligand present. Differential scanningfluorimetry (DSF), utilizes a fluorescent dye that interacts withhydrophobic residues in a protein, producing a fluorescent signal thatis monitored over a temperature range where the protein unfoldingoccurs. As the protein unfolds, hydrophobic residues that were buried inthe protein's three dimensional structure become exposed allowing newinteractions with the dye molecule. In theory, a ligand that binds theprotein's native state will increase the protein stability resulting ina higher temperature needed to unfold or denature the protein, resultingin a higher melting temperature, T_(m). Limitations of the thermal shiftassay are that the ΔT_(m) values represent relative binding efficiencyand the values do not indicate how the ligand is binding, or forinstance if the compound inhibits enzyme activity. Also, solubility ofligands in the buffered solution are limiting on the concentrations thatcan be tested, as seen in all assays. Thermal shift assays are veryuseful as a screening method and a means of evaluation of ligands forhighly soluble fragments.

Compounds synthesized were tested in a thermal shift assay againstMycobacterium paratuberculosis IspD (MpIspD) and Escherichia coli IspD(EcIspD) as well as Mycobacterium tuberculosis IspD (MpIspD) andLycopersicon esculentum IspE (LeIspE) and the results are shown in FIG.50 and Table 1.

Overall, the results from the thermal shift data show that all compoundsare showing a relatively low change in melting point. HGN-207 showed a0.8° C. positive shift for MpIspD and HGN-211, HGN-208, HGN-241, andHGN-221 all had equal shifts or higher. HGN-211 used had atrifluoromethyl substituent on the oxadiazole ring and a 0.3° C.increase in melting point over HGN-207. A higher shift was seen withHGN-208 in which the substituent on the oxadiazole ring was a phenyl andwhen the phenyl was extended out by one methylene unit in HGN-221, thethermal shift was 1.5° C., the highest obtained against MpIspD. InEclspD, many thermal shifts were negative, which may mean that thecompounds were destabilizing the enzyme or interacting more with the dyemolecules. Due to the large number of negative values, this data doesnot show a clear correlation to MpIspD thermal shift data. Similarresults were seen with LeIspE data shown in Table 1. The thermal shiftdata for MtIspD shows the hit molecule, HGN-207, at a 1.5° C. positiveshift and HGN-239, HGN-252, HGN-214, and HGN-336 all have equal orhigher shifts. The highest shift seen was HGN-214 with a 1.8° C.positive shift in which the substituent is a 4-(trifluoromethyl)phenyl.Benzothiazole and benzimidazole analogs of HGN-214 did not show positiveshifts as high however.

IspE Expression and Purification of E. coli and B. thailandensis

The Ec IspE gene in parent vector pET14b (obtained from Dr. Karen Meyerslab). The Bt IspE gene was obtained from the SSGCID (ButhA. 00725.a. A1)within the parent vector AVA0421. Each parent vector introduces the sameantibiotic resistance and restriction sites as the IspD. Also, similarto IspD expression, the vectors were transformed into BL21(DE3)competent cells. Both DNA sequences were verified through University ofChicago CRC-DNA sequencing facility. The transformed cells were grown onLB agar plates with 100 μg/ml of amp for selection of the expressionplasmid, and a single colony was chosen to inoculate a five mL of LB(100 μg/ml amp). This five mL sample was incubated overnight at 37° C.while shaking at 235 RPM. A 50 mL LB (100 μg/ml amp) subculture wasinoculated with the overnight culture. This subculture was grown at 37°C. with shaking (235 RPM) until reaching an OD₆₀₀ in a range of 0.5-0.8.Once at mid-log, the cell culture is induced with IPTG to a finalconcentration of 1.0 mM. The temperature is dropped to 20° C. and thecells were grown for 16 hours while shaking at 235 RPM. After the 16hours, the bacterial cells were pelleted using centrifugation at 8,000RPM for 15 minutes and stored at −20° C.

Purification for both species of IspE was performed as described forIspD. Affinity chromatography was executed using a HisTrap HP nickelaffinity IMAC column (GE Healthcare Life Sciences) with a BioLogic LP(Bio-Rad Life Sciences) fast protein liquid chromatography (FPLC)system. Size exclusion chromatography performed with a Superdex 75HiLoad 26/60 size exclusion column (GE Healthcare Life Sciences). Buffersystems were identical for all enzymes as described herein.

Thermal Stability Assays

All thermal stability assays were completed identical to IspD usingcircular dichroism and differential scanning fluorimetry.

Enzyme Inhibition Determination with Kinase-Glo® Luminescent Assay

Similar to the EnzChek® enzymatic assay, determination of activity isfundamental for evaluation of inhibitor. CDP-ME undergoesphosphorylation by IspE and ATP, leading to the product CDP-MEP. TheKinase-Glo® assay follows the consumption of the reactant ATP. The ATPin conjunction with oxygen and beetle luciferin are catalyzed byluciferase with the cofactor of magnesium to produce oxyluciferin, AMP,carbon dioxide, pyrophosphate, and creates a luminescence signal. Thisluminescence is directly related to the amount of ATP within thereaction, as the reaction progresses, the ATP is consumed and the amountof luminescence is decreased.

All Kinase-Glo® reactions were run using the following conditions (allconditions are described in final concentrations): 60 mM NaCl, 5 mMMgCl₂, 20 mM HEPES, 1 mM DTT, 5% DMSO (for compound suspension), 0.01%BSA, 200 μM CDP-ME, 40 μM ATP, and either Ec IspE and Bt IspE. All butthe substrate CDP-ME are combined and incubated with the compounds for10 minutes, before the addition of substrate and incubation (45 minutes,with shaking, room temperature). After incubation, the Kinase-Glo®reagent was added in equal final volume and allowed to develop for 10minutes. Luminescence was recorded via a Synergy 2 plate reader.

Reactions involving the compounds and Kinase-Glo® reagent are performedto verify that the compounds are not interfering with luciferase enzymein the reagent itself. The oxadiazole series are similar to knownluciferase inhibitors, which tend to resemble the substrate, beetleluciferin. Most of the oxadiazole series, the beetle luciferin and theresembling known inhibitor (D-luciferin, 6-methyl ether) all have thecore structure of benzothiazole. However, the difference is that insteadof the oxadiazole, the inhibitor and substrate have a dihydrothiazole.

ITC Binding Studies

All ITC experiments were performed under the same conditions asdescribed herein. Compounds were dissolved in DMSO and brought toconcentration with the dialysis buffer, with a maximum DMSOconcentration of 10% by volume which was matched between ligand andenzyme, and centrifuged at 10,000 RPM for 10 minutes. The dialysisbuffer was used for all sample dilutions to ensure buffers matchingbetween the titrant and titrate, thus minimizing the excess heat due tobuffer mismatch. ITC experiments were performed with IspE in the celland ligand in the syringe. The initial injection volume was 2 μL(omitted) followed by 27 10 μL injections, every 240 seconds. Sampleswere run at 25° C., at pH 7.4, with a stirring speed of 307 RPM.Dilution runs were performed to allow determination of background heatscaused by solution mismatch and instrument noise. Dilution runs wereperformed with ligand (at the same concentration as in the experimentrun) in the syringe titrated into the dialysis buffer in the cell. Datawere fit using OriginPro 7 with the ITC add-on by the manufacturer.

Expression of IspE for Both E. coli and B. thailandensis Results

IspE enzymes were purified using a nickel affinity column with a 20-500mM gradient elution using imidazole. Fractions associated with the UVabsorbance peak in the chromatogram, verified with a NanoDrop 2000cspectrophotometer (Thermo Scientific), were combined and concentrated to10 mL using a spin concentrator. The resulting concentrate was furtherpurified using a size exclusion chromatography column (Superdex 75HiLoad 26/60), and fractions related to the UV absorbance peak of IspDwere collected, combined, and concentrated.

Melting Point Determination of IspE

Circular Dichroism was used to determine the thermal stability of Ec andBt IspE. Wavelength scans were run before the thermal melt to get abaseline reading which confirmed the mainly alpha helical secondarystructure of the proteins. Full scans were repeated post-thermal melt tocharacterize the protein's reversibility. Both species show loss ofsecondary structure which may suggest the thermal unfolding to benon-reversible under the conditions of the experiment. The thermalstability of each of the proteins was observed at a single wavelengthover a temperature range of 25-100° C. The melting temperature (T_(m))was determined to be approximately 50° C. for Ec IspE and 46° C. for BtIspE, which will be used as reference temperatures for further thermalshift assays.

Thermal Shift Results

Thermal stability assay performed as described herein. The range ofmelting point shifts for pyrazolopyrimidines are from −1.5° C. to +4° C.The range for the oxadiazole series was much more diverse with a rangeof −0.5° C. to +6.5° C.

Kinase-Glo® Enzymatic Assay Results

Pyrazolopyrimidines and oxadiazoles were evaluated in an enzymatic assayagainst both E. coli and B. thailandensis. The oxadiazole series, whichwere screened, have a structure similar to the luciferin reactant in theKinase-Glo® reagent. To verify that the compounds are not interactingwith the luciferase, involved in the secondary reaction which convertsthe beetle luciferin to oxyluciferin, a separate reaction is run sansenzyme.

This assay is performed with all reagents without enzyme and compoundsconcentration was 250 μM. This verification is necessary to ensure theinhibition values of the compounds are accurate. The assay resultsrevealed only a few compounds in the pyrazolopyrimidine series possessedinhibition activity with IC5₀ values in the high micromolar range. Theoxadiazole series showed a greater range in activity from low to highmicromolar with the greatest activity being from compound HGN-0333 at3±0.3 μM for E. coli and 28±6.5 μM for B. thailandensis.

Binding Studies of Bt IspE with HGN-0333

ITC studies were performed to observe the dissociation constant alongwith thermodynamic properties of the oxadiazole compound HGN-0333 withBt IspE. The experiment showed a binding stoichiometry of 0.903 whichwould indicate that for each monomer there is one molecule of HGN-0333.The ΔH° of HGN-0333 showed to have a value of −8.6 kcal/mol and anentropic contribution of −TΔS of −17.8. K_(D app) of HGN-0333 was shownto be in the nanomolar range.

2. Lettuce Leaf Assay

The lettuce leaf assay is a qualitative assay used to determine therelative effectiveness of the test compounds at inhibition ofgermination of seeds and root development as well as causing bleachingof leaves. Isoprenoid synthesis is vital in plants as isoprene-productgibberellins are a class of growth hormones in plants responsible forstimulating cell elongation and division. In addition, some carotenoidsare involved in photosynthesis as components of the electron transportchain. MEP pathway inhibitors have the potential to prevent synthesis ofthese necessary cellular components and growth hormones resulting ininhibition of germination and root development as well as causingchlorosis, or bleaching in the leaves. The lettuce leaf assay, exploresthe effects of compounds as potential MEP pathway inhibitors byevaluating the effect on bleaching, root development, and germination oflettuce seeds, which all rely on isoprenoid biosynthesis. This assaydoes not directly identify MEP pathway inhibitors but is used as asupplemental assay to enzymatic inhibition assays. The assay is aqualitative assay and the effects are given rankings from 0 (no effect)to 5 (complete inhibition or bleaching). In this assay, compounds aredissolved in a volatile organic solvent and applied to filter paper in apetri dish. The solvent is allowed to evaporate and lettuce seeds areapplied followed by the addition of 5 mL of water to obtain thenecessary concentrations. Seeds are allowed to grow and they areobserved on day 3 and day 4 for evaluation. The results are shown inTable 2.

From the lettuce leaf assay it is shown that HGN-207 did show inhibitionof germination slightly and a fair amount of chlorosis. At aconcentration of 1.0 mM, HGN-242 and HGN-223 showed the greatestactivity at inhibiting germination of the lettuce seeds. A 4 in thiscase represents 80% of the seeds did not show any growth. HGN-214, whichhad an electron withdrawing group at the 4 position on the phenyl ringshowed good inhibition of germination at 1.0 mM but not to the extent aswhen there is an electron donating group seen in HGN-242 and HGN-223.Many compounds were tested at a maximum concentration of 0.3 mM due tosolubility issues. In these cases, DMSO stock solutions were made anddiluted to a maximum of 1% DMSO in water. Because of this lowerconcentration and low water solubility, the data may not berepresentative of the compounds ability to inhibit MEP pathway enzymesas they may not have been able to pass into the lettuce seeds.

3. ISPD Assay (FIG. 55A-55F and FIG. 56A-56F)

Compounds were tested against IspD from Escherichia coli and Plasmodiumfalciparum. The assay measured activity through monitoring phosphatelevels released by IspD enzyme over time using conditions publishedpreviously. The assay utilized the EnzCheck phosphate assay kit toquantify phosphate released by IspD. Reactions were done in a 96-wellplate with each well containing a final volume of 50 μL. Concentrationsof reagents were as follows: 100 mM NaCl, 25 mM Tris at pH 7.0, 7.5 mMMgCl₂, 1 mM dithiothreitol, 1 U/mL purine nucleoside phosphorylase, and0.1 U/mL yeast inorganic pyrophosphatase, 50 μM CTP, 500 μM MEP, 0.1%DMSO, and 0.2 mM 2-Amino-6-mercapto-7-methyl puring riboside. Reactionswith EclspD used 2 nM EclspD and reactions with PfIspD used 50 nMPfIspD. Reactions involved pre-warming the enzyme and buffers to 37° C.with different inhibitor concentrations followed by the addition of thepurified MEP substrate. Absorbance was measured at 360 nm over time andvalues were used to create regression analysis curves from three trialsand values were converted to (μM MEP)(μM enzyme)⁻¹s⁻¹ using a standardphosphate curve. These values were then used to identify percentactivity compared to enzyme activity without the inhibitor. Results areindicated.

Results of the IspD assay show that most compounds tested were inactiveup to 100 μM against E. coli IspD and P. falciparum IspD enzymes asindicated in the bar graph of FIG. 51. The only compound that showedsignificant activity was HGN-252 with a cyclopentyl substituent at both12.5 and 100 μM concentrations. From the raw data, it appears thatHGN-213 and HGN-214 from both E. coli IspD and P. falciparum IspD showsome activity. The plot from E. coli IspD for HGN-214 had significantnoise but there is a clear decrease in enzyme activity at higherconcentrations. A significant decrease in activity was seen as well withHGN-213 and HGN-214 against P. falciparum IspD with IC₅₀ valuesapproaching 100 μM. (FIG. 51A-51B)

4. Enzymatic Inhibition Assay from University of Hamburg

Compounds were subjected to a photometric enzymatic assay to test forinhibition of the plant enzymes Arabidopsis thaliana IspD and IspF, andIspE from Lycopersicon esculentum. The assay done was by using a coupledreaction scheme shown in FIGS. 52-54.

The procedure for AtIspD enzymatic inhibition assay, was performed bymaking an assay mixture of 100 mM Tris hydrochloride at pH 8.0 with 10mM MgCl₂, 1 mM dithiothreitol, 2.5 mM potassium phosphoenol pyruvate,2.1 mM CTP, 2 mM ATP, 1 mM of (2R,3S)-2,3-dihydroxy-4-oxopentylphosphate, 460 μM NADH, 1.1% (v/v) DMSO, 0.4 U IspE, 1 U lactatedehydrogenase, 1 U pyruvate kinase and 2.4 mU IspD in 180 μL ofsolution. Solutions were incubated at 27° C. and monitoredphotometrically. By preparing the assay mixture with a large excess ofIspE compared to IspD, any inhibition of IspE by the test inhibitorwould be insignificant compared to IspD and therefore the rate of thereaction is determined by IspD activity.

The IspE assay conditions were very similar to the IspD assay in whichthe assay mixture was composed of 100 mM Tris hydrochloride at pH 8.0with 10 mM MgCl₂, 1 mM dithiothreitol, 2.5 mM potassium phosphoenolpyruvate, 2 mM ATP, 1 mM of 4-Diphosphocytidyl-2-C-methylerythritol, 460μM NADH, 1.1% (v/v) DMSO, 2.4 mU IspE, 1 U lactate dehydrogenase, 1 Upyruvate kinase in 180 μL of solution. Solutions were incubated at 27°C. and monitored photometrically.

The IspF assay mixture conditions were as follows: 100 mM Trishydrochloride at pH 8.0 with 10 mM MgCl₂, 1 mM dithiothreitol, 2.5 mMpotassium phosphoenol pyruvate, 2 mM ATP, 1 mM of 4-diphosphocytidyl-2Cmethylerythritol 2-phosphate, 460 μM NADH, 1.1% (v/v) DMSO, 0.5 U CMPkinase, 2.4 mU IspF, 1 U lactate dehydrogenase, 1 U pyruvate kinase in180 μL of solution. Solutions were incubated at 27° C. and monitoredphotometrically.

Results of the assay against AtIspD, LeIspE, and AtIspF are summarizedin Table 3.

Data from Table 3 was the most compelling data obtained for the compoundlibrary as it provided clear IC₅₀ values with associated error. In Table3, the parent compound HGN-207 did not show any measurable activityagainst AtIspD, LeIspE, or AtIspF. No significant activity increase wasseen until HGN-254 showed an IC₅₀ of 322 μM against AtIspD. Changing thecyclohexyl group to a phenyl increased activity against LeIspE to 165 μMand no significant change in activity for AtIspD. The most potentcompounds for the indole series were those in which the phenyl ring wassubstituted at the 4 position with an electron donating group such asmethyl or methoxy as seen with HGN-242 and HGN-223, both having low 200μM IC₅₀ values against AtIspD and low 100 μM IC₅₀ values against LeIspE.Changing the indole ring of HGN-242 and HGN-223 to a benzothiazole ringin HGN-338 and HGN-336 showed a significant loss in activity. Thecompounds HGN-213 and HGN-214, which showed activity in Dr. AudreyOdom's assay, had relatively low activity in the University of Hamburgenzymatic inhibition assay. HGN-213 had an IC₅₀ value of 490 μM againstAtIspD and the benzothiazole analog HGN-339 showed an increase inactivity to 158 μM and changing the benzothiazole ring to abenzimidazole in HGN-421 showed an even better activity at an IC₅₀ 88μM.

Of particular interest are compounds HGN-333 and HGN-340. HGN-333 showedactivity against LeIspE at 31 μM and AtIspD at 151 μM as well as AtIspFat 291 μM. This compound has significant error associated with thesevalues due to solubility issues of the compound. The pseudilin-likecompound HGN-340 had the best activity against AtIspD at 50 μM.

5. Antibacterial Assay

The results from an antibacterial assay are from a primary screening ofcompounds against nine different strains of bacteria. The test organismsincluded both gram positive and gram negative bacteria. Thegram-positive bacteria included Bacillus cereus (BC), Micrococcus luteus(ML), Mycobacterium smegmatis (MS), Corynebacterium xerosis (CX), andCorynebacterium pseudodiphtheriae (CP). Gram negative bacteria includedBurkholderia thailandensis (BT), Klebsiella pneumonia (KP), Escherichiacoli (EC), and Pseudomonas aeruginosa (PA). The primary screening wasperformed by streaking pure cultures of the test organisms onto separateagar plates and then isolating a single colony from each plate andinoculating it into a 5 mL liquid broth. This broth was incubatedovernight at 37° C. Each test organism was spread on a plate toinoculate the entire surface and edges. Five compounds were tested oneach plate by adding dry compound to the plate, after which 5 μL of 5%DMSO was added to the spots. Plates were then incubated at 37° C.overnight and checked for bacterial growth and zones of inhibitionaround the compounds. Test compounds were given scores from 0-3depending on their ability to inhibit growth. The results are shown inTable 4.

SAR Evaluation of Topliss Tree Set

Using FOL7082 (HGN-207) as a hit molecule in the fragment based STD-NMRscreening, a fragment growing technique, utilizing the Topliss treeapproach, was used. Three assays evaluated compound activity againstenzymes of a variety of species. The thermal shift assay measured howwell compounds stabilized the enzyme. Both enzyme assays provided IC₅₀values, with plant enzymes tested by one IspD from both Escherichia coliand Plasmodium falciparum at Washington University. Also, two assaytested compound activity in vivo, which were done utilizing nine strainsof bacteria and in a lettuce leaf assay.

Evaluating the SAR of the compound library is difficult as there is noX-ray crystal structure of IspD or IspE in complex with FOL7082(HGN-207) to evaluate important interactions between the compound andthe enzyme. A Topliss tree approach was taken to make deliberate changesand determine which were beneficial based on the activity seen in assayresults. One enzymatic assay produced the most substantial resultsbecause the IC₅₀ values for each compound were determined for IspD,IspE, and IspF, and provide a direct comparison between compounds.Beginning with the original hit, HGN-207, the IC₅₀ was determined to behigher than 1000 μM. By substituting on the oxadiazole ring at the 5position with alkyl groups to increase the lipophilicity by substitutinga methyl group in place of hydrogen and growing the fragment with ethyl,isopropyl, t-butyl. Unfortunately, the upper limit to this assay was1000 μM of compound. Most of the substitutions, whether made to increaselipophilicity or affect the electronics of the molecule, were at orabove the 1000 μM limit while the cyclohexyl substitution had an IC₅₀ of322 μM against A. thaliana IspD. This suggests that a bulky lipophilicgroup is beneficial.

Moving from a cyclohexyl group to a phenyl, the activity against IspDwas not significantly affected but now there is a significant increasein activity against IspE, going from an IC₅₀ greater than 1000 μM to 165μM. The three dimensional structure of HGN-208 is very planar withlittle flexibility. By extending the phenyl ring out in HGN-221 andHGN-215, IspD activity was lost and activity against IspE wasdiminished. Going back to HGN-208, analogs were made by addingsubstituents to the phenyl ring at different positions. Adding achlorine to the 3 position on the phenyl ring resulted in a loss ofactivity against IspD but no significant change against IspE. When achlorine was moved to the 4 position on the phenyl ring, activity wasgained back but against IspD, the activity was not as high as theunsubstituted ring. Activity against IspE was also lower in the 4-chlorosubstituted phenyl compared to unsubstituted HGN-208. When the chlorineis changed to a trifluoromethyl, IspD activity is lost, indicating thata strongly electron withdrawing group negatively affects the activityfor IspD. Substituting these electron withdrawing groups with electrondonating groups at the 4 position such as a methyl group with HGN-242and methoxy group with HGN-223, activity was significantly enhanced forboth IspD and IspE. IspD activity was in the low 200 μM range and IspEactivity was in the low 100 μM range for both HGN-242 and HGN-223.

Using the four, 4-substituted compounds that showed the best activityagainst IspD and IspE, analogs were made to determine how changing theindole ring to a different heterocycle might affect the activity. Abenzothiazole series was first explored. HGN-339, the benzothiazoleanalog of HGN-213, had an increase in IspD activity, but IspE activitywas lost. For the other benzothiazole analogs, activity wassignificantly lower compared to the indole series. A benzimidazoleseries was also explored with analogs of HGN-213 and HGN-214. Thebenzimidazole series showed a significant increase in activity formostly IspD. The activity increased from 490 to 88 μM in moving fromHGN-213 to HGN-421, and >1000 to 251 μM moving from HGN-214 to HGN-422.In both, there was not a significant change in IspE activity.

Compounds HGN-333, HGN-334, and HGN-335, were all made because theyshowed antibacterial activity in published reports with no indication ofmechanism of action, but with structural similarities to activecompounds. They were explored for activity against MEP pathway enzymes.Of particular interest is HGN-333, which showed significant activityagainst all three enzymes in the University of Hamburg assays, however,due to solubility issues, there was some concern about the trueactivity. However, it is noteworthy, as it did show a significant effecton IspE.

HGN-340 does not fit in with the typical structure of any previouslytested compounds, but the Witschel group reported pseudilin compoundsthat showed good activity against IspD by acting as an allostericinhibitor of the enzyme. HGN-340 was made as an analog of the pseudilincompound reported. In the enzymatic assay herein, it had the bestactivity of all the compounds tested, with an IC₅₀ value of 50 μM.

Comparing the results from the two enzymatic assays, the data isconsistent with compounds HGN-213 and HGN-214 being the most active ofthe compounds tested. It can be noted that of the alkyl substitutedcompounds, HGN-252 showed the best inhibitory activity at both 12.5 and100 μM concentrations and when that is compared to results fromUniversity of Hamburg data, it shows an inconsistency as HGN-254 hadbetter activity in the University of Hamburg assay over HGN-252. Thismay be a result of differences in sequence homology between E. coli andA. thaliana IspD enzymes.

In the thermal shift assay, the results are more challenging to explainin terms of activity of the compound. The thermal shift data wascollected against M. tuberculosis IspD and L. esculentum IspE enzymes.In the thermal shift assay against IspD, HGN-214 had the highestpositive shift in melting point of 1.8° C., but HGN-214 was inactiveagainst AtIspD in one assay. However, the other assay showed thatHGN-214 had the best activity against E. coli IspD. Differences betweenspecies may result in the inconsistent activities, especially if thecompounds are acting as allosteric inhibitors as there is a likely ahigher variability in sequence between enzymes outside of the activesite. The thermal shift assay results provide insight into the relativebinding affinity of ligands and while many produced a shift in themelting point of IspD, these changes are quite small, most likelyindicating compounds are weak binders. In the thermal shift assay forIspE, most compounds produced a negative thermal shift. In theory, if amolecule binds to the enzyme, it should stabilize it producing apositive thermal shift. The negative shifts for LeIspE indicate thatmany compounds may have destabilized the enzyme, acting as a denaturantto some extent. LeIspE was used in both the thermal shift and one assayso the results should be comparable, but oddly, the only compound thatshowed a positive thermal shift was found to be inactive against LeIspE,and HGN-250 had a significant negative thermal shift with −2.75° C., butwas inactive in one assay could have a significant effect. However,HGN-422 had the largest negative shift at −3.25° C. and showed fairlydecent activity as well with an IC₅₀ value of 267 μM. HGN-422 wasn't themost potent compound against LeIspE in one assay.

In vivo assays are of particular interest when looking for potentialantibacterial or herbicidal agents. The antibacterial preliminaryscreening, showed interesting results for a few compounds that showconsistency with the current SAR. Most compounds tested showed noinhibition of bacterial growth amongst the nine species tested. From theUniversity of Hamburg assay, HGN-421 was of particular interest becauseit had an IC₅₀ against IspD of 88 μM. HGN-421 and HGN-422 both showedslight antibacterial activity against Burkholderia thailandensis andMycobacterium smegmatis. HGN-333, was moderately effective againstPseudomonas aeruginosa while HGN-335 was slightly effective. HGN-340,which had an activity of 50 μM in the University of Hamburg assay, wasmoderately effective against P. aeruginosa as well. This shows there issome consistency between the enzymatic assay and the antibacterialassay, but likely due to poor solubility of compounds in water, many maynot have been able to enter bacterial cells, resulting in loweractivities in the antibacterial screening.

A second in vivo assay was used to evaluate efficacy of compounds atinhibiting germination and root development in lettuce seeds or causingbleaching of the leaves. Results from this assay revealed that theparent compound HGN-207 caused significant bleaching and inhibition ofgermination at 1.0 mM and 0.5 mM. HGN-209 resulted in some inhibition ofroot development and germination also at 1.0 mM and 0.5 mM. Furthersubstitutions resulted in a loss of activity against root developmentbut many caused some inhibition in root germination, such as HGN-250that caused a significant decrease in germination at 1.0 mM and mostothers resulting in slight inhibition.

Materials and Methods

Design of Compound Library

1. Topliss Tree Approach

Typically, the drug design process is a lengthy task in developinganalogs of an initial hit molecule to ultimately reach a highly potentdrug. Often times, thousands of compounds may be generated to optimizeinteractions of the hit molecule with the target. In the 1960s and 70s,the Hansch methods were developed to provide a rational approach forquantitative structure-activity relationships (QSAR) in a compoundlibrary. These methods helped determine which properties havesignificant biological activity input. Some properties of varioussubstituents involve the hydrophobicity constant of that substituent, π,relative to hydrogen, the Hammett substituent constant, which gives ameasure of the electronic properties of the substituent (electronwithdrawing or electron donating), denoted as σ, and Taft's stericfactor, denoted as E_(S). Using these factors, a mathematical derivationof the relationship with the level of activity of analogs could allowfor development of analogs likely to display enhanced activity.

Professor J. G. Topliss developed a non-mathematical operation scheme inthe 1970s that utilized the Hansch method parameters to progress rapidlyto more potent compounds in a series of analogs. The non-mathematicalscheme presented by Topliss became known as the Topliss tree (FIGS. 12and 13). This stepwise method typically begins with an unsubstitutedphenyl compound and a 4-chloro substituted phenyl analog or anunsubstituted parent compound in which aliphatic groups are added inplace of hydrogen. In the case of phenyl substitutions, the 4-chlorosubstituent represents a +π value and +σ value because it is bothlipophilic and electron withdrawing. The 4-chloro substituent iscompared to the unsubstituted phenyl compound in potency and classifiedas less potent, equally potent, or more potent. If the compound is morepotent, it can likely be attributed to a beneficial +π, +σ, or acombined effect. Therefore, a 3, 4-dichloro analog is synthesized next,as the +π and +σ effects are larger and could possibly lead to greaterpotency. However, if 4-chloro was equally as potent as the unsubstitutedphenyl, then it is most likely attributed to a beneficial +π effect andbut offset by a detrimental +σ effect, because typically increasinglipophilic character is better for small molecule binding affinity butthe electron withdrawing effects may have been too large. Therefore, thenext appropriate compound to make would be a 4-methyl substituted phenylbecause this has a lipophilic character, +π, but it is also electronreleasing, −σ. If the 4-chloro analog is significantly less potent thanthe unsubstituted phenyl, then it may be due to a steric interactionfrom the 4-substitution, or the activity is enhanced by a −π or −σsubstituent (hydrophilic or electron donating). The most likely causefor the decrease in potency is due to a need for a −σ effect, sofollowing the 4-chloro substitution, the next analog would be 4-methoxydue to its enhanced electron donating effect and only slightly enhancedhydrophilic character. A similar rational is used for the rest of thedecision tree to designate different substituents on the phenyl ring. Asimilar scheme has also been applied to series with aliphatic side chainsubstitutions as well.

2. Design of Chemical Series

Without X-ray crystal structures of MpIspD and MaIspE with FOL7082bound, it is difficult to make deliberate changes to the fragment toincrease the binding efficiency. Therefore, the Topliss tree approachwas taken utilizing the fragment FOL7082 as the core to develop a seriesof compounds to test for enzyme inhibition activity. The simplest methodof generating a series of analogs was to determine which areas of themolecule would most suitable to change.

Two areas of variability are the two heterocyles, the indole and the1,3,4-oxadiazole. Typically, a fragment screening will identifyfragments that have low binding affinity but high ligand efficiency.Therefore, the core fragment was kept constant throughout the analogseries to maintain the high efficiency of the binding and the fragmentwas expanded to introduce new binding sites. Based on chemical synthesisof FOL7082, the most ideal area of variability was to substitute variousfunctional groups on the 1, 3,4-oxadiazole ring. Therefore, startingwith the unsubstituted fragment, the following Topliss tree schemes weredeveloped. (FIG. 14A-14B)

3. Pseudilin-Type Ispd Inhibitor

In addition to the Topliss tree set of compounds, literature searchesrevealed similar compounds reported to have low micromolar IC₅₀ values.These compounds, evaluated using Arabidopsis thaliana IspD, were shownto bind to an allosteric binding site, possibly promoted by the highlevel of halogenation. While there were certain similarities to FOL7082,there are not enough to imply that they are binding in the same sites.The compound of interest is a pentabromopseudilin showing an IC₅₀ valueof 13±2 μM without a metal added but 1.4±0.2 μM with 40 μM Cd²⁺ added.The x-ray crystal structure with the pentabromopseudilin shows thehalogen bonding and metal-ion interactions in AtIspD. Thepentabromopseudilin compound synthetic route is 7 steps.

A simpler analog of the pentabromopseudlin compound may result in asimilar activity with fewer steps. The analog chosen was the following:

This compound could be synthesized in 3 steps by utilizing theFischer-Indole synthesis. This compound is an analog of both thefragment hit FOL7082 and of the pentabromopseudilin. By changing the1,3,4-oxadiazole ring of FOL7082 to a 2,4-dibromo phenol, this compoundrepresents a single change analog of FOL7082 as shown below.

The x-ray crystal structure of pentabromopseudilin shows the pyrrolenitrogen coordinating with Cd²⁺, and this is likely due to the fact thatthe bromines on the pyrrole ring increase the acidity of the N—H moiety.The hydroxyl group on the phenol is also much more acidic by placingbromines ortho and para to it. The coordination to the Cd²⁺ is dependenton these acidities. In the FOL7082 analog, the brominated phenol allowsfor the hydroxyl acidity but without any indole substitutions, thecoordination between the indole N—H and metal will likely be much weakerthan the pseudilin compound and activity will likely be lower because ofthis if it binds the same way to the enzyme. However, the indole groupallows for a larger amount of possible variability as it can be easilysubstituted, providing a starting point for a new set of analogs.

Preparation of Compounds

1. Indole Series (FIGS. 16-38)

To a round bottom flask add 0.005 moles of ethyl indole-2-carboxylatewith 20 mL ethanol. Add 0.5 mL hydrazine hydrate (50-60%). Refluxsolution for 6 hours. Allow solution to cool to room temperature andallow product to precipitate out by placing round bottom flask in an icebath. Filter product and recrystallize in ethanol. Yield 77%.

2-(1H-indol-2-yl)-1,3,4-oxadiazole (HGN-207)

To a dry, 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(211.6 mg, 1.208 mmol) and 15 mL formic acid. The resulting solution wasstirred at reflux for 6 hours. Excess formic acid was removed underreduced pressure and the resulting solid was washed with cold ethanol,filtered, and dried solid. Yield of N′-formyl-1H-indole-2-carbohydrazidewas 240.6 mg (98.0%). 214.8 mg (1.057 mmol) of resulting solid wasdissolved and refluxed in 6 mL POCl₃ for 2 hours. The resulting reactionmixture was cooled to room temperature and poured over crushed ice.Saturated sodium bicarbonate solution was added slowly while stirringuntil solution was slightly basic (pH=8). Solid product was filtered andwashed with cold ethanol and dried. Product was recrystallized withethanol to produce 2-(1H-indol-2-yl)-1,3,4-oxadiazole (195.7 mg, 59%yield). ¹H NMR 300 MHz (DMSO-d₆) 12.28 (1H, s), 9.36 (1H,$), 7.68 (1H,d, J=8.0 Hz), 7.49 (1H, d, J=8.3 Hz), 7.27 (1H, t, J=7.2 Hz), 7.23 (1H,s), 7.11 (1H, t, J=7.0); ¹³C NMR 300 MHz (DMSO-d₆): 159.45, 154.33,138.21, 127.69, 124.71, 121.96, 121.38, 120.82, 112.75, 105.49;(cESI-MS) m/z [M]⁺ calcd for C₁₀H₈N₃O⁺ 186.07, found 186.2; m.p.205-207° C.

2-(1H-indol-2-yl)-5-phenyl-1,3,4-oxadiazole (HGN-208)

To a dry, 100 mL round bottom flask was added benzoic acid (146.6 mg,1.200 mmol) and 1H-indole-2-carbohydrazide (175.2 mg, 1.000 mmol)followed by 6 mL POCl₃ and the resulting mixture was refluxed for 6 h.The reaction mixture was cooled to room temperature and excess POCl₃ wasremoved under reduced pressure. Cold water was added to reaction vesselfollowed with saturated sodium bicarbonate solution until pH wasslightly basic (pH=8). Product was filtered and dried and purified viacolumn chromatography using a gradient of ethyl acetate in hexanestarting with 1:5 ethyl acetate:hexane. Product was then recrystallizedin ethanol to yield 2-(1H-indol-2-yl)-5-phenyl-1,3,4-oxadiazole (57.4mg, 22% yield). ¹H NMR 300 MHz (DMSO-d₆) 12.34 (1H, s), 8.14 (2H, dd,J=2.19, 5.52), 7.72-7.65 (4H, m), 7.52 (1H, d, J=8.3 Hz), 7.34 (1H, s),7.28 (1H, t, J=7.6 Hz), 7.12 (1H, t, J=7.6 Hz); ¹³C NMR 300 MHz(DMSO-d₆):163.83, 159.87, 138.29, 132.51, 129.93, 127.79, 127.10,124.75, 123.74, 121.96, 121.51, 120.85, 112.73, 105.62; (cESI-MS) m/z[M]⁺ calcd for C₁₆H₁₂N₃O⁺ 262.10, found 262.2; m.p 254-257° C.

2-(1H-indol-2-yl)-5-methyl-1,3,4-oxadiazole (HGN-209)

To a dry, 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(175.2 mg, 1 mmol) and 15 mL of glacial acetic acid. Resulting mixturewas refluxed for 6 h. Excess acetic acid was removed under reducedpressure to yield 247.6 mg crude product, which was then recrystallizedin ethanol. Product was filtered, dried, and added to a clean, 100 mLround bottom flask. 6 mL POCl₃ was added and solution was refluxed for 2hours under nitrogen. Excess POCl₃ was removed under reduced pressureand crushed ice was added to reaction vessel. The mixture was pouredinto a beaker and a saturated sodium bicarbonate solution was addedslowly until pH was slightly basic (pH=8). Product was filtered, dried,and collected to yield 162.7 mg of product, which was then purifiedusing column chromatography with a gradient starting at 5% methanol indichloromethane to yield 2-(1H-indol-2-yl)-5-methyl-1,3,4-oxadiazole(89.0 mg, 45% yield). ¹H NMR 300 MHz (DMSO-d₆) 12.21 (1H, s), 7.66 (1H,d, J=7.98 Hz), 7.47 (1H, d, J=8.16 Hz), 7.25 (1H, t, J=7.28 Hz), 7.14(1H,$), 7.09 (1H, t, J=7.41 Hz), 2.60 (3H, s); ¹³C NMR 300 MHz(DMSO-d₆): 163.77, 159.78, 138.09,127.71, 124.50, 121.85, 121.73,120.72, 112.68, 104.80, 11.06; (cESI-MS) m/z [M]⁺ calcd for C₁₁H₁₀N₃O⁺200.08, found 200.2; m.p. 232-234° C.

2-(1H-indol-2-yl)-5-(trifluoromethyl)-1,3,4-oxadiazole (HGN-211)

To a 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(175.2 mg, 1 mmol) and 6 mL POCl₃ followed by trifluoroacetic acid(171.0 mg, 1.5 mmol). The resulting mixture was refluxed for 6 h. Thereaction mixture was poured over crushed ice and saturated sodiumbicarbonate solution was added slowly until the mixture was slightlybasic (pH=8). Product was filtered and dried followed by purificationvia column chromatography using 10% ethyl acetate in hexane as theeluting solvent to yield2-(1H-indol-2-yl)-5-(trifluoromethyl)-1,3,4-oxadiazole (164.8 mg, 65.1%yield). ¹H NMR 300 MHz (DMSO-d₆) 12.50 (1H, s), 7.71 (1H, d, J=7.92 Hz),7.51 (1H, d, J=8.31 Hz), 7.40 (1H, s), 7.31 (1H, t, J=7.21 Hz), 7.14(1H, t, J=6.93 Hz); ¹³C NMR 300 MHz (DMSO-d₆): 161.70, 153.97, 138.66,127.58, 125.49, 122.30, (C—F q 121.15, 118.55, 114.97, 111.51, J=241Hz), 119.87, 112.95, 107.52; m.p. 182-184° C.

2-(1H-indol-2-yl)-5-(methoxymethyl)-1,3,4-oxadiazole (HGN-212)

To a 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(175.2 mg, 1 mmol) and 6 mL POCl₃ followed by methoxyacetic acid (135.1mg, 1.5 mmol). The mixture was refluxed for 2 h. The reaction mixturewas poured over crushed ice and saturated sodium bicarbonate solutionwas added slowly until mixture was slightly basic (pH=8). Solid productwas filtered and dried followed by purification via columnchromatography using a 1:2 mixture of ethyl acetate in hexane to yield2-(1H-indol-2-yl)-5-(methoxymethyl)-1,3,4-oxadiazole (94.1 mg, 41%). ¹HNMR 300 MHz (DMSO-d₆) 12.30 (1H, s), 7.68 (1H, d, J=8.01 Hz), 7.48 (1H,d, J=8.28 Hz), 7.27 (1H, t, J=7.63 Hz), 7.22 (1H,$), 7.11 (1H, t, J=7.00Hz), 4.75 (2H, s), 3.41 (3H, s); ¹³C NMR 300 MHz (DMSO-d₆): 163.24,160.43, 136.25, 127.69, 124.75, 121.97, 121.29, 120.84, 112.76, 105.54,63.59, 58.79; (cESI-MS) m/z [M]⁺ calcd for C₁₂H₁₂N₃O₂ ⁺ 230.09, found230.2; m.p. 152-156° C.

2-(4-chlorophenyl)-5-(1H-indol-2-yl)-1,3,4-oxadiazole (HGN-213)

To a 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(175.2 mg, 1.0 mmol) and 6 mL POCl₃ followed by 4-chlorobenzoic acid(234.6 mg, 1.5 mmol). The resulting mixture was heated to reflux untilfully dissolved and then the temperature was reduced to 60° C. for 2 h.The resulting reaction mixture was the poured over crushed ice andsaturated sodium bicarbonate solution was added until the mixture wasslightly basic (pH=8). Solid product was filtered and dried followed bypurification via column chromatography using a gradient of 12% ethylacetate in hexane to 60% ethyl acetate in hexane. Product crystallizedin the test tubes and was collected and dried to yield2-(4-chlorophenyl)-5-(1H-indol-2-yl)-1,3,4-oxadiazole (110.9 mg, 38%yield). ¹H NMR 300 MHz (DMSO-d₆) 12.35 (1H, s), 8.20-8.08 (2H, m),7.80-7.65 (3H, m), 7.51 (1H, d, J=8.28 Hz), 7.34 (1H,$), 7.28 (1H, t,J=7.60 Hz), 7.12 (1H, t, J=7.13 Hz); ¹³C NMR 300 MHz (DMSO-d₆): 163.09,159.99, 138.30, 137.22, 130.12, 128.88, 127.77, 124.82, 122.64, 121.99,121.38, 120.88, 112.73, 105.77; (cESI-MS) m/z [M]⁺ calcd forC₁₆H₁₁ClN₃O⁺ 296.06, found 296.1; m.p. 265-270° C.

2-(1H-indol-2-yl)-5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazole(HGN-214)

To a 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(175.2 mg, 1.0 mmol) and 6 mL POCl₃ followed by4-(trifluoromethyl)benzoic acid (228.1 mg, 1.2 mmol). The resultingmixture was heated to reflux until fully dissolved and then thetemperature was reduced to 60° C. for 2 h. The resulting reactionmixture was poured over crushed ice and saturated sodium bicarbonatesolution was added slowly until the mixture was slightly basic (pH=8).Solid product was filtered, dried, and recrystallization in ethanol withdecolorizing carbon. Product was collected filtered and dried to yield2-(1H-indol-2-yl)-5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazole (103.4mg, 31% yield). ¹H NMR 300 MHz (DMSO-d₆) 12.39 (1H, s), 8.34 (2H, d,J=7.8 Hz), 8.04 (2H, d, J=8.4 Hz), 7.72 (1H, d, J=7.8 Hz), 7.52 (1H, d,J=8.4 Hz), 7.38 (1H, s), 7.29 (1H, t, J=8.0 Hz), 7.10 (1H, t, J=8.0 Hz);¹³C NMR 300 MHz (DMSO-d₆): 162.82, 160.36, 138.36, 132.19, 131.17, (C—Fq 128.18, 126.94, 124.92, 122.44, J=143.5 Hz) 127.91, 127.77, 127.54,126.89, 122.04, 121.26, 120.92, 112.77, 106.03; (cESI-MS) m/z [M]⁺ calcdfor C₁₇H₁₁F₃N₃O⁺ 330.09, found 330.3; m.p. 258-260° C.

2-(1H-indol-2-yl)-5-phenethyl-1,3,4-oxadiazole (HGN-215)

To a 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(175.2 mg, 1.0 mmol), hydrocinnamic acid (180.2 mg, 1.2 mmol) and 6 mLPOCl₃. The mixture was heated to reflux until solid was fully dissolved,followed by a reduction in heat to 65° C. for 4 h under nitrogen. Theresulting mixture was poured over crushed ice and saturated sodiumbicarbonate solution was added slowly until solution was slightly basic(pH=8). Solid was filtered, collected, and dried to produce 567.8 mg ofcrude product. Product was purified via column chromatography using agradient of ethyl acetate in hexane starting at 8% ethyl acetate andmoving to 66% ethyl acetate. Further purification was done viarecrystallization in ethanol to yield2-(1H-indol-2-yl)-5-phenethyl-1,3,4-oxadiazole (169.3 mg, 44% yield). ¹HNMR 300 MHz (DMSO-d₆) 12.22 (1H, s), 7.67 (1H, d, J=7.89 Hz), 7.48 (1H,t, J=8.04 Hz), 7.31-7.18 (6H, m), 7.14 (1H,$), 7.10 (1H, t, J=7.49 Hz),3.28 (2H, t, J=7.68 Hz), 3.13 (2H, t, J=7.49 Hz); ¹³C NMR 300 MHz(DMSO-d₆): 166.02, 159.71, 140.29, 138.12, 128.89, 128.82, 127.71,126.83, 124.56, 121.87, 121.65, 120.76, 112.69, 104.97, 32.04, 26.85;(cESI-MS) m/z [M]⁺ calcd for C₁₈H₁₆N₃O⁺ 290.13, found 290.3; m.p.161-164° C.

2-benzyl-5-(1H-indol-2-yl)-1,3,4-oxadiazole (HGN-221)

To a 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(175.2 mg, 1.0 mmol), phenylacetic acid (163.4 mg, 1.2 mmol) and 6 mLPOCl₃. The mixture was heated to reflux until all solid was dissolvedfollowed by a reduction in heat to 65° C. for 4 h under nitrogen. Theresulting mixture was cooled to room temperature and poured over crushedice and saturated sodium bicarbonate solution was added until solutionwas slightly basic (pH=8). Solid was filtered, collected, and dried.Crude product was purified via recrystallization in ethanol to yield2-benzyl-5-(1H-indol-2-yl)-1,3,4-oxadiazole (157.3 mg, 57%). ¹H NMR 300MHz (DMSO-d₆) 12.22 (1H, s), 7.65 (1H, d, J=7.95 Hz), 7.47 (1H, d,J=8.19 Hz), 7.43-7.28 (5H, m), 7.25 (1H, t, J=7.58 Hz), 7.14 (1H, s),7.09 (1H, t, J=7.44 Hz), 4.38 (2H, s); ¹³C NMR 300 MHz (DMSO-d₆):165.44, 160.08, 138.16, 134.90, 129.40, 129.25, 127.73, 127.68, 124.60,121.89, 121.49, 120.76, 112.70, 105.16, 31.21; (cESI-MS) m/z [M]⁺ calcdfor C₁₇H₁₄N₃O⁺276.11, found 276.3; m.p. 211-215° C.

To a 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(350.4 mg, 2.0 mmol), 4-methoxybenzoic acid (365.2 mg, 2.4 mmol) and 12mL POCl₃. The mixture was heated to reflux until all solid was dissolvedfollowed by a reduction in heat to 85° C. for 4 h under nitrogen. Theresulting mixture was cooled to room temperature and poured over crushedice and saturated sodium bicarbonate solution was added until solutionwas slightly basic (pH=8). Solid was filtered, collected, and dried.Crude product was purified via recrystallization in ethanol to yield2-(1H-indol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazole (111.2 mg, 19.1%yield). ¹H NMR 300 MHz (DMSO-d₆) 12.32 (1H, s), 8.07 (2H, d, J=8.7 Hz),7.70 (1H, d, J=9 Hz), 7.51 (1H, d, J=8.7 Hz), 7.35-7.09 (5H, m), 3.87(3H, s); ¹³C NMR 300 MHz (DMSO-d₆): 163.78, 162.58, 159.38, 138.21,128.97, 127.80, 124.63, 121.90, 121.65, 120.80, 116.03, 115.40, 112.70,105.30, 56.03; m.p. 243-246° C.

(mass spec data not available due to low solubility in hplc grademethanol and acetonitrile)

2-(tert-butyl)-5-(1H-indol-2-yl)-1,3,4-oxadiazole (HGN-0239)

To a 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(175.2 mg, 1.0 mmol), pivalic acid (122.6 mg, 1.2 mmol) and 6 mL POCl₃.The mixture was heated to reflux until all solid was dissolved followedby a reduction in heat to 65° C. for 6 h under nitrogen. The resultingmixture was cooled to room temperature and poured over crushed ice andsaturated sodium bicarbonate solution was added until solution wasslightly basic (pH=8). Solid was filtered, collected, and dried. Crudeproduct was purified via recrystallization in ethanol to yield2-(tert-butyl)-5-(1H-indol-2-yl)-1,3,4-oxadiazole (77.9 mg, 32%). ¹H NMR300 MHz (DMSO-d₆) 12.20 (1H, s), 7.66 (1H, d, J=7.5 Hz), 7.48 (1H, d,J=8.1 Hz), 7.25 (1H, t, J=7.7 Hz), 7.18 (1H, s), 7.09 (1H, t, J=7.5 Hz),1.44 (9H, s); ¹³C NMR 300 MHz (DMSO-d₆): 172.57, 159.71, 138.10, 127.71,124.52, 121.85, 121.71, 120.73, 112.68, 105.02, 32.52, 28.30; (cESI-MS)m/z [M]⁺ calcd for C₁₄H₁₆N₃O⁺ 242.13, found 242.3; m.p. 163-168° C.

2-(1H-indol-2-yl)-5-isopropyl-1,3,4-oxadiazole (HGN-240)

To a 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(175.2 mg, 1.0 mmol), isobutyric acid (105.7 mg, 1.2 mmol) and 6 mLPOCl₃. The mixture was heated to reflux to dissolve reactants followedby a reduction in heat to 70° C. for 24 h under nitrogen. The resultingmixture was cooled and poured over crushed ice and saturated sodiumbicarbonate solution was added slowly until solution was slightly basic(pH=8). Solid was filtered, collected, and dried to produce 233.1 mg ofcrude product. Product was extracted with ethyl acetate and water. Theorganic layer was collected and dried over anhydrous sodium sulfate. Thesolution was transferred to a 250 mL round bottom flask and solvent wasremoved under reduced pressure. Product was purified viarecrystallization in acetone and product was filtered and dried to yield2-(1H-indol-2-yl)-5-isopropyl-1,3,4-oxadiazole (122.1 mg, 54% yield). ¹HNMR 300 MHz (DMSO-d₆) 12.21 (1H, s), 7.66 (1H, d, J=7.50), 7.47 (1H, d,J=7.74 Hz), 7.25 (1H, t, J=7.20 Hz), 7.17 (1H, s), 7.09 (1H, t, 7.19),3.29 (1H, m), 1.38 (6H, d, J=6.42 Hz); ¹³C NMR 300 MHz (DMSO-d₆):170.45, 159.66, 138.10, 127.71, 124.50, 121.84, 121.73, 120.72, 112.67,104.94, 26.11, 20.19; (cESI-MS) m/z [M]⁺ calcd for C₁₃H-₁₄N₃O⁺ 228.11,found 228.2; m.p. 164-170° C.

2-(3-chlorophenyl)-5-(1H-indol-2-yl)-1,3,4-oxadiazole (HGN-241)

To a 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(175.2 mg), 3-chlorobenzoic acid (187.9 mg, 1.2 mmol) and 6 mL POCl₃.The mixture was heated to reflux with stirring until all solid was fullydissolved followed by a reduction in heat to 65° C. for 4 h undernitrogen. The resulting mixture was cooled to room temperature andpoured over crushed ice and saturated sodium bicarbonate solution wasadded slowly until solution was slightly basic (pH=8). Solid wasfiltered, collected, dried, and purified via recrystallization inacetone to yield 2-(3-chlorophenyl)-5-(1H-indol-2-yl)-1,3,4-oxadiazole(74.5 mg, 25% yield). ¹H NMR 300 MHz (DMSO-d₆) 12.35 (1H, s), 8.16 (1H,s), 8.10 (1H, d, J=7.41 Hz), 7.78-7.64 (3H, m), 7.52 (1H, d, J=8.49 Hz),7.38 (1H, s), 7.29 (1H, t, J=7.14), 7.12 (1H, t, J=7.47 Hz); ¹³C NMR 300MHz (DMSO-d₆): 162.67, 160.12, 138.30, 134.58, 132.27, 131.97, 127.78,126.64, 125.74, 125.70, 124.86, 122.01, 121.32, 120.90, 112.73, 105.94;(cESI-MS) m/z [M]⁺ calcd for C₁₆H₁₁ClN₃O⁺ 296.06, found 296.2; m.p.202-207° C.

2-(1H-indol-2-yl)-5-(p-tolyl)-1,3,4-oxadiazole (HGN-242)

To a 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(175.2 mg, 1.0 mmol), p-toluic acid (163.4 mg, 1.2 mmol) and 6 mL POCl₃.The resulting mixture was heated to reflux with stirring until all solidwas fully dissolved followed by a reduction in heat to 65° C. for 4 hunder nitrogen. The mixture was cooled to room temperature and pouredover crushed ice and saturated sodium bicarbonate solution was addedslowly until solution was slightly basic (pH=8). Solid was filtered,collected, dried, and purified via recrystallization in acetone to yield2-(1H-indol-2-yl)-5-(p-tolyl)-1,3,4-oxadiazole (101.2 mg, 37% yield). ¹HNMR 300 MHz (DMSO-d₆) 12.41 (1H, s), 8.03 (2H, d, J=7.98), 7.69 (1H, d,J=8.46), 7.52 (1H, d, J=8.49 Hz), 7.46 (2H, d, J=7.98 Hz), 7.31 (1H, s),7.27 (1H, t, J=7.76 Hz), 7.11 (1H, t, J=7.25 Hz), 2.42 (3H, s); ¹³C NMR300 MHz (DMSO-d₆): 163.91, 159.62, 142.69, 138.25, 130.47, 127.77,127.07, 124.65, 121.91, 121.54, 120.96, 120.81, 105.41, 21.64; (cESI-MS)m/z [M]⁺ calcd for C₁₇K₄N₃O⁺ 276.11, found 276.2; m.p. 228-234° C.

3-(5-(1H-indol-2-yl)-1,3,4-oxadiazol-2-yl)-N,N-dimethylaniline (HGN-249)

To a 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(175.2 mg, 1.0 mmol), 3-(dimethylamino)benzoic acid (165.2 mg, 1.0 mmol)and 6 mL POCl₃. The resulting mixture was heated to reflux with stirringuntil all solid was fully dissolved followed by a reduction in heat to70° C. for 6 h under nitrogen. The resulting mixture was cooled to roomtemperature and poured over crushed ice and saturated sodium bicarbonatesolution was added slowly until solution was slightly basic (pH=8).Solid was filtered, collected, dried, and purified via recrystallizationin ethanol to yield3-(5-(1H-indol-2-yl)-1,3,4-oxadiazol-2-yl)-N,N-dimethylaniline (136.6mg, 45%). Product did not completely dissolve in hot ethanol and theproduct was still impure. Solubility issues prevented properpurification and pure product was not isolated.

2-ethyl-5-(1H-indol-2-yl)-1,3,4-oxadiazole (HGN-250)

To a 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(175.2 mg, 1.0 mmol), propionic acid (88.9 mg, 1.2 mmol) and 6 mL POCl₃.The resulting mixture was heated to reflux until all solid was fullydissolved followed by a reduction in heat to 75° C. for 6 h undernitrogen. The mixture was cooled to room temperature and poured overcrushed ice and saturated sodium bicarbonate solution was added slowlyuntil solution was slightly basic (pH=8). Solid product was filtered,collected, and dried followed by extraction with ethyl acetate andwater. The organic layer was dried over anhydrous sodium sulfate.Organic layer was then transferred to a round bottom flask and solventwas removed under reduced pressure. Product was purified viarecrystallization in ethanol to yield2-ethyl-5-(1H-indol-2-yl)-1,3,4-oxadiazole (117.8 mg, 55% yield). ¹H NMR300 MHz (DMSO-d₆) 12.21 (1H, s), 7.66 (1H, d, J=7.44 Hz), 7.48 (1H, d,J=7.62 Hz), 7.25 (1H, t, J=6.99 Hz), 7.15 (1H, s), 7.10 (1H, t, J=7.40Hz), 3.02-2.86 (2H, m), 1.35 (3H, t, J=7.02 Hz); ¹³C NMR 300 MHz(DMSO-d₆): 167.59, 159.69, 138.10, 127.72, 124.50, 121.85, 121.75,120.72, 112.68, 104.86, 18.84, 10.89; m.p. 138-143° C.

(mass spec data not available due to low solubility in hplc grademethanol and acetonitrile)

2-cyclobutyl-5-(1H-indol-2-yl)-1,3,4-oxadiazole (HGN-251)

To a 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(175.2 mg, 1.0 mmol), cyclobutanecarboxylic acid (120.1 mg, 1.2 mmol)and 6 mL POCl₃. The resulting mixture was heated to reflux until allsolid was fully dissolved, followed by a reduction in heat to 75° C. for6 h under nitrogen. The mixture was cooled and poured over crushed iceand saturated sodium bicarbonate solution was added slowly untilsolution was slightly basic. Solid was filtered, collected, and dried toproduce 294.3 mg of crude product. Purification was done viarecrystallization in acetone to yield2-cyclobutyl-5-(1H-indol-2-yl)-1,3,4-oxadiazole (127.0 mg, 53% yield).¹H NMR 300 MHz (DMSO-d₆) 12.21 (1H, s), 7.66 (1H, d, J=7.95 Hz), 7.48(1H, d, J=8.25 Hz), 7.24 (1H, t, J=7.62 Hz), 7.17 (1H, s), 7.09 (1H, t,J=7.49 Hz), 3.85 (1H, quin., J=8.45 Hz), 2.43 (4H, q, J=8.07 Hz),2.18-1.95 (2H, m); ¹³C NMR 300 MHz (DMSO-d₆): 168.69, 159.71, 138.12,127.73, 124.50, 121.84, 121.75, 120.72, 112.68, 104.96, 30.04, 26.79,18.78; (cESI-MS) m/z [M]⁺ calcd for C₁₄H₁₄N₃O⁺240.11, found 240.2; m.p.169-173° C.

2-cyclopentyl-5-(1H-indol-2-yl)-1,3,4-oxadiazole (HGN-252)

To a 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(175.2 mg, 1.0 mmol), cyclopentanecarboxylic acid (137.0 mg, 1.2 mmol)and 6 mL POCl₃. The resulting mixture was heated to reflux until allsolid was fully dissolved, followed by a reduction in heat to 75° C. for6 hours under nitrogen. The mixture was cooled and poured over crushedice and saturated sodium bicarbonate solution was added slowly untilsolution was slightly basic (pH=8). Solid was filtered, collected, anddried. Purification was done via recrystallization in ethanol to yield2-cyclopentyl-5-(1H-indol-2-yl)-1,3,4-oxadiazole (164.8 mg, 54% yield).¹H NMR 300 MHz (DMSO-d₆); ¹³C NMR 300 MHz (DMSO-d₆): 169.16, 159.24,137.62, 127.23, 123.97, 121.32, 121.29, 120.21, 112.17, 104.41, 35.13,30.49, 25.01; (cESI-MS) m/z [M]⁺ calcd for C₁₅H₁₆N₃O⁺ 254.13, found254.2; m.p. 150-154° C.

2-(3,4-dichlorophenyl)-5-(1H-indol-2-yl)-1,3,4-oxadiazole (HGN-253)

To a 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(175.2 mg, 1.0 mmol), 3,4-dichlorobenzoyl chloride (209.5 mg, 1.0 mmol)and 6 mL POCl₃. The mixture was heated to reflux until all solid wasfully dissolved, followed by a reduction in heat to 70° C. for 5 hoursunder nitrogen. Excess POCl₃ was removed under reduced pressure and themixture was poured onto crushed ice and saturated sodium bicarbonatesolution was added slowly until solution was slightly basic (pH=8).Solid product was filtered, collected, and extracted with ethyl acetateand water. Organic layer was dried over anhydrous sodium sulfate,collected, and solvent was removed under reduced pressure to produce358.8 mg of crude product. Product was purified via recrystallization inacetone to yield2-(3,4-dichlorophenyl)-5-(1H-indol-2-yl)-1,3,4-oxadiazole (96.2 mg, 28%yield). Product was still impure after recrystallization and furtherpurification would yield too little product to continue.

2-cyclohexyl-5-(1H-indol-2-yl)-1,3,4-oxadiazole (HGN-254)

To a 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(175.2 mg, 1.0 mmol), cyclohexanecarboxylic acid (153.8 mg, 1.2 mmol)and 6 mL POCl₃. The mixture was heated to reflux until all solid isdissolved, followed by a reduction in heat to 65° C. for 6 hours undernitrogen. The mixture was cooled and poured over crushed ice andsaturated sodium bicarbonate solution was slowly added until solutionwas slightly basic (pH=8). Solid product was filtered, collected, anddried to produce 417.7 mg of crude product. Product was purified viarecrystallization in acetone to yield2-cyclohexyl-5-(1H-indol-2-yl)-1,3,4-oxadiazole (154.3 mg, 49% yield).¹H NMR 300 MHz (DMSO-d₆) 12.18 (1H, s), 7.66 (1H, d, J=7.92 Hz), 7.48(1H, d, J=8.22 Hz), 7.25 (1H, t, J=7.62 Hz), 7.15 (1H, s), 7.09 (1H, t,J=7.49 Hz), 3.05 (1H, tt, J=3.69 Hz, 10.82 Hz), 2.15-2.03 (2H, m),1.84-1.73 (2H, m), 1.73-1.52 (3H, m), 1.52-1.20 (3H, m); ¹³C NMR 300 MHz(DMSO-d₆): 168.88, 158.98, 137.62, 127.23, 123.99, 121.33, 121.27,120.22, 112.18, 104.41, 34.11, 29.57, 25.16, 24.65; (cESI-MS) m/z [M]⁺calcd for C₁₆H₁₈N₃O⁺ 268.14, found 268.2; m.p. 187-193° C.

To a 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(175.2 mg, 1.0 mmol), [1,1′-biphenyl]-3-carboxylic acid (237.9 mg, 1.2mmol) and 6 mL POCl₃. The mixture was heated to reflux until all solidis dissolved, followed by a reduction in heat to 75° C. for 6 hoursunder nitrogen. The mixture was cooled and poured over crushed ice andsaturated sodium bicarbonate solution was slowly added until solutionwas slightly basic (pH=8). Solid product was filtered, collected, anddried to produce 307.7 mg of crude product. Product was purified viarecrystallization in ethanol to yield2-([1,1′-biphenyl]-3-yl)-5-(1H-indol-2-yl)-1,3,4-oxadiazole (95.4 mg,28% yield). ¹H NMR 300 MHz (DMSO-d₆) 12.33 (1H, s), 8.38 (1H, s), 8.15(1H, d, J=9 Hz), 7.97 (1H, d, J=8.1 Hz), 7.85-7.68 (4H, m), 7.60-7.43(4H, m), 7.40 (1H, s), 7.29 (1H, t, J=7.8 Hz), 7.13 (1H, t, J=7.4 Hz);¹³C NMR 300 MHz (DMSO-d₆): 163.29, 159.47, 141.34, 138.92, 137.81,130.26, 130.16, 129.11, 128.15, 127.33, 126.90, 125.57, 124.64, 124.28,123.95, 121.47, 121.01, 120.37, 112.23, 105.32; m.p. 222-226° C.

(mass spec data not available due to low solubility in hplc grademethanol and acetonitrile)

To a 100 mL round bottom flask was added 1H-indole-2-carbohydrazide(175.2 mg, 1.0 mmol), [1,1′-biphenyl]-4-carboxylic acid (237.9 mg, 1.2mmol) and 6 mL POCl₃. The mixture was heated to reflux until all solidis dissolved, followed by a reduction in heat to 75° C. for 6 hoursunder nitrogen. The mixture was cooled and poured over crushed ice andsaturated sodium bicarbonate solution was slowly added until solutionwas slightly basic (pH=8). Solid product was filtered, collected, anddried to produce 320.8 mg of crude product. Product was purified viarecrystallization in ethanol to yield2-([1,1′-biphenyl]-4-yl)-5-(1H-indol-2-yl)-1,3,4-oxadiazole (102.4 mg,30% yield). ¹H NMR 300 MHz (DMSO-d₆) 12.34 (1H, s), 8.23 (1H, d, J=8.4Hz), 7.98 (1H, d, J=8.4 Hz), 7.90-7.61 (4H, m), 7.60-7.42 (4H, m), 7.37(1H, s), 7.29 (1H, t, J=7.8 Hz), 7.13 (1H, t, J=8.0 Hz); ¹³C NMR 300 MHz(DMSO-d₆): 143.88, 139.24, 138.30, 130.42, 129.62, 129.54, 128.87,128.09, 127.73, 127.37, 127.28, 124.78, 122.61, 121.98, 121.53, 120.88,112.74, 105.66; m.p. 258-260° C.

(mass spec data not available due to low solubility in hplc grademethanol and acetonitrile)

2. Benzothiazole Series. (FIGS. 39-46)

Preparation of benzo[d]thiazole-2-carbohydrazide (FIG. 39)

To a 250 mL round bottom flask was added ethylbenzothiazole-2-carboxylate (1.0363 g, 5.0 mmol). 20 mL ethanol and 2 mLhydrazine hydrate were added followed by heating the solution to refluxfor 6 hours. The reaction mixture was cooled to room temperature andsolid precipitate was filtered, collected, and recrystallized in ethanolto yield benzo[d]thiazole-2-carbohydrazide (943.4 mg, 89.1% yield).

2-(benzo[d]thiazol-2-yl)-5-((2,4-dichlorophenoxy)methyl)-1,3,4-oxadiazole(HGN-333)

To a 250 mL round bottom flask was addedbenzo[d]thiazole-2-carbohydrazide (193.2 mg, 1.0 mmol),2,4-dichlorophenoxyacetic acid (442.1 mg, 2.0 mmol), and 10 mL POCl₃.The resulting mixture was refluxed for 6 hours. The solution was cooledto room temperature and poured over crushed ice. The solution was leftuntil precipitate formed, after which saturated sodium bicarbonatesolution was added until solution was slightly basic. Product wasextracted with ethyl acetate and the organic layer was collected anddried over anhydrous sodium sulfate. Organic layer was transferred to aclean 250 mL round bottom flask and solvent was removed under reducedpressure to yield 699.4 mg of crude product which was recrystallized inethanol to produce 256.3 mg of product. Further purification was donevia column chromatography using 25% ethyl acetate in hexane to yield2-(benzo[d]thiazol-2-yl)-5-((2,4-dichlorophenoxy)methyl)-1,3,4-oxadiazole(171.9 mg, 46% yield). ¹H NMR 300 MHz (CDCl₃) 8.31 (1H, dd, J=2.03, 6.33Hz), 8.25 (1H, dd, J=2.03, 6.72 Hz), 7.73-7.61 (3H, m), 7.49-7.39 (2H,m), 5.72 (2H, s); ¹³C NMR 300 MHz (CDCl₃): 162.85, 161.40, 153.25,152.00, 150.00, 135.57, 130.58, 128.25, 127.86, 127.68, 127.43, 124.98,124.92, 122.02, 116.10, 61.28; (cESI-MS) m/z [M]⁺ calcd forC₁₆H-₁₀Cl₂N₃O₂S⁺ 377.99, found 377.1; m.p. 163-165° C.

2-(benzo[d]thiazol-2-yl)-5-(3,5-dinitrophenyl)-1,3,4-oxadiazole(HGN-334)

To a 250 mL round bottom flask was addedbenzo[d]thiazole-2-carbohydrazide (193.2 mg, 1.0 mmol),3,5-dinitrobenzoic acid (424.2 mg, 2.0 mmol), and 10 mL POCl₃. Resultingsolution was refluxed for 6 hours. Reaction mixture was cooled to roomtemperature and poured over crushed ice. The solution was allowed to situntil product precipitated from solution, after which saturated sodiumbicarbonate solution was slowly added until the solution was slightlybasic (pH=8). Product was extracted using ethyl acetate. The organiclayer was collected and dried over anhydrous sodium sulfate. Organiclayer was then transferred to a clean 250 mL round bottom flask andsolvent was removed under reduced pressure to yield 755.4 mg of crudeproduct. Product was recrystallized in ethanol to yield2-(benzo[d]thiazol-2-yl)-5-(3,5-dinitrophenyl)-1,3,4-oxadiazole (234.4mg, 64%). ¹H NMR 300 MHz (CDCl₃) 9.42 (2H, s), 9.27 (1H, s), 8.32 (1H,d, J=8.10 Hz), 8.08 (1H, d, J=7.62 Hz), 5.72 (2H, s), 7.72-7.61 (2H, m);¹³C NMR 300 MHz (DMSO-d₆): 162.28, 161.42, 149.42, 149.29, 135.72,128.00, 127.69, 127.01, 126.56, 124.99, 122.12, 121.57; (cESI-MS) m/z[M]⁺ calcd for C₁₅H₈N₅O₅S⁺ 370.02, found 371.0; m.p. 180-185° C.

(E)-2-(benzo[d]thiazol-2-yl)-5-styryl-1,3,4-oxadiazole (HGN-335)

To a 250 mL round bottom flask was addedbenzo[d]thiazole-2-carbohydrazide (193.2 mg, 1.0 mmol), trans cinnamicacid (177.8 mg, 1.2 mmol), and 10 mL POCl₃. Resulting solution wasrefluxed for 6 hours. Reaction mixture was allowed to cool to roomtemperature and poured over crushed ice. The solution was allowed to situntil product precipitated out, after which saturated sodium bicarbonatesolution was slowly added until solution was slightly basic (pH=8).Product was extracted with ethyl acetate and the organic layer wascollected and dried over anhydrous sodium sulfate. Organic layer wastransferred to a clean 250 mL round bottom flask and solvent was removedunder reduced pressure to produce(E)-2-(benzo[d]thiazol-2-yl)-5-styryl-1,3,4-oxadiazole (296.1 mg, 92%yield). ¹H NMR 300 MHz (CDCl₃) 8.33-8.24 (1H, m), 8.28 (1H, dd, J=1.71,9.42 Hz), 7.91-7.84 (2H, m), 7.80 (1H, s), 7.72-7.62 (2H, m), 7.53 (1H,s), 7.51-7.42 (3H, m); ¹³C NMR 300 MHz (CDCl₃): 165.59, 159.59, 153.35,150.87, 141.24, 135.49, 134.50, 130.45, 129.11, 127.77, 127.40, 127.29,124.69, 121.99, 108.97; (cESI-MS) m/z [M]⁺ calcd for C₁₇H₁₂N₃OS⁺ 306.07,found 306.3; m.p. 202-204° C.

2-(benzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazole (HGN-336)

To a 250 mL round bottom flask was addedbenzo[d]thiazole-2-carbohydrazide (193.2 mg, 1.0 mmol), 4-methoxybenzoicacid (304.3 mg, 2.0 mmol), and 10 mL POCl₃. Resulting mixture wasrefluxed for 6 hours. Reaction mixture was allowed to cool to roomtemperature and poured over crushed ice and allowed to sit until productprecipitated out and saturated sodium bicarbonate solution was addeduntil solution was slightly basic (pH=8). Product was extracted withethyl acetate and the organic layer was collected and dried overanhydrous sodium sulfate. Organic layer was transferred to a clean 250mL round bottom flask and solvent was removed under reduced pressure toyield 318.4 mg of crude product. Product was recrystallized in ethanolto produce 2-(benzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazole(105.6 mg, 34% yield). ¹H NMR 300 MHz (CDCl₃) 8.27 (1H, d, J. 8.04 Hz),8.21 (2H, d, J=8.64 Hz), 7.66-7.54 (3H, m), 7.07 (2H, d, J=8.64 Hz),3.93 (3H, s); ¹³C NMR 300 MHz (CDCl₃): 165.92, 163.01, 159.80, 153.38,151.05, 135.44, 129.45, 127.25, 127.19, 124.62, 121.95, 115.47, 114.65,55.52; (cESI-MS) m/z [M]⁺ calcd for C₁₆H₁₂N₃O₂S⁺ 310.07, found 310.2;m.p. 167-169° C.

2-(benzo[d]thiazol-2-yl)-5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazole(HGN-337)

To a 250 mL round bottom flask was addedbenzo[d]thiazole-2-carbohydrazide (386.5 mg, 2.0 mmol),4-(trifluoromethyl)benzoic acid (380.2 mg, 2.0 mmol), and 10 mL POCl₃.Mixture was refluxed for 6 hours and allowed to cool to roomtemperature. Solution was poured over crushed ice and allowed to situntil product precipitated out. Saturated sodium bicarbonate solutionwas added slowly until solution is slightly basic (pH=8). Product wasextracted with ethyl acetate and the organic layer was collected anddried over anhydrous sodium sulfate. Solution was transferred to a clean250 mL round bottom flask and solvent was removed under reduced pressureto produce 238.9 mg of crude product. Product was recrystallized inethanol to yield2-(benzo[d]thiazol-2-yl)-5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazole(197.1 mg, 28% yield). ¹H NMR 300 MHz (CDCl₃) 8.41 (2H, d, J=8.10 Hz),8.29 (1H, d, J=7.23 Hz), 8.05 (1H, d, J=7.83 Hz), 7.86 (2H, d, J=8.22Hz), 7.65 (1H, td, J=1.48, 7.73 Hz), 7.59 (1H, td, J=1.41, 7.38 Hz); ¹³CNMR 300 MHz (CDCl₃): 164.67, 160.69, 153.34, 150.41, 135.56, 134.72,134.26, 133.82, 133.43, 127.90, 127.61, (C—F q 127.42, 125.29, 124.80,122.05, J=134.1 Hz), 126.29, 126.24; (cESI-MS) m/z [M]⁺ calcd forC₁₆H₉F₃N₃OS⁺ 348.04, found 348.2; m.p. 145-148° C.

2-(benzo[d]thiazol-2-yl)-5-(p-tolyl)-1,3,4-oxadiazole (HGN-338)

To a 250 mL round bottom flask was addedbenzo[d]thiazole-2-carbohydrazide (386.5 mg, 2.0 mmol), p-toluic acid(272.3 mg, 2.0 mmol), and 10 mL POCl₃. Resulting solution was refluxedfor 6 hours, allowed to cool to room temperature, and pour over crushedice. The reaction mixture was allowed to sit until product precipitatedout after which saturated sodium bicarbonate solution was added untilsolution was slightly basic (pH=8). Product was extracted with ethylacetate and the organic layer was collected and dried with anhydroussodium sulfate. Solution was transferred to a clean 250 mL round bottomflask and solvent was removed followed by recrystallization in ethanolto yield 2-(benzo[d]thiazol-2-yl)-5-(p-tolyl)-1,3,4-oxadiazole (157.3mg, 27% yield). ¹H NMR 300 MHz (DMSO-d₆) 8.28 (1H, d, J=7.41 Hz), 8.16(2H, d, J=6.6 Hz), 8.04 (1H, d, J=7.62 Hz), 7.63 (1H, td, J=1.47, 7.68Hz), 7.57 (1H, td, J=1.34, 7.43 Hz), 2.48 (3H, s); ¹³C NMR 300 MHz(DMSO-d₆): 165.66, 160.14, 153.25, 151.25, 143.51, 135.30, 130.63,128.17, 127.97, 127.49, 124.62, 123.42, 120.38, 21.68; (cESI-MS) m/z[M]⁺ calcd for C₁₆H₁₂N₃OS⁺ 294.07, found 294.1; m.p. 155-160° C.

2-(benzo[d]thiazol-2-yl)-5-(4-chlorophenyl)-1,3,4-oxadiazole (HGN-339)

To a 250 mL round bottom flask was addedbenzo[d]thiazole-2-carbohydrazide (386.5 mg, 2.0 mmol), 4-chlorobenzoicacid (313.1 mg, 2.0 mmol), and 10 mL POCl₃. Resulting solution wasrefluxed for 6 hours, allowed to cool to room temp and pour over crushedice. The reaction mixture was allowed to sit until product precipitatedout, after which saturated sodium bicarbonate solution was slowly addeduntil solution was slightly basic (pH=8). Product was extracted withethyl acetate and the organic layer was collected and dried overanhydrous sodium sulfate. Organic layer was collected in a clean 250 mLround bottom flask and solvent was removed under reduced pressure toyield 344.6 mg crude product. Product was recrystallized in ethanol toyield 2-(benzo[d]thiazol-2-yl)-5-(4-chlorophenyl)-1,3,4-oxadiazole(185.6 mg, 30% yield). ¹H NMR 300 MHz (DMSO-d₆) 8.27 (1H, d, J=7.95 Hz),8.21 (2H, d, J=8.61 Hz), 8.04 (1H, d, J=6.96 Hz), 7.67-7.55 (4H, m); ¹³CNMR 300 MHz (CDCl₃): 165.10, 160.32, 153.33, 150.61, 138.94, 135.50,129.63, 128.82, 127.49, 127.34, 124.73, 122.02, 121.50; (cESI-MS) m/z[M]⁺ calcd for C₁₅H₉ClN₃OS⁺ 314.02, found 314.1; m.p. 161-163° C.

3. Pseudilin-Like Series (FIG. 47)

2,4-dibromo-6-(1H-indol-2-yl)phenol (HGN-340)

To a 250 mL round bottom flask was added by 2′-hydroxyacetophenone (5.0g, 36.7 mmol) and 60 mL of methanol. This solution was cooled to 0° C.in an ice bath. N-bromosuccinimide (19.6 g, 110.2 mmol) was added slowlywith stirring to the solution on ice. The solution was brought up toroom temperature, sealed, and allowed to stir overnight. The solutionwas poured into 100 mL water and allowed to sit producing a light yellowprecipitate. The product was filtered, collected, and recrystallized inethanol to yield 1-(3,5-dibromo-2-hydroxyphenyl)ethanone (2.2940 g, 21%yield). 1-(3,5-dibromo-2-hydroxyphenyl)ethanone (456.8 mg, 1.55 mmol)was added to a 100 mL round bottom flask with 10 mL ethanol and stirred.Phenylhydrazine (0.15 mL, 1.5 mmol) was added with 5 drops of glacialacetic acid and mixture was refluxed at 80° C. for 1 hour. The solutionwas cooled to room temperature and the solvent is evaporated off underreduced pressure to yield 598.1 mg crude product((E)-2,4-dibromo-6-(1-(2-phenylhydrazono)ethyl)phenol). 10 mL ofmethanesulfonic acid was added to(E)-2,4-dibromo-6-(1-(2-phenylhydrazono)ethyl)phenol, which was notpurified from the previous step. The mixture was heated at 100° C. for30 minutes and then poured over crushed ice and neutralized with 1 MNaOH solution. The product was extracted with ethyl acetate, dried overanhydrous sodium sulfate, and concentrated. Purification was done usinga gradient of ethyl acetate in hexane to yield2,4-dibromo-6-(1H-indol-2-yl)phenol (342.1 mg, 60% yield, 12.7% yieldoverall). ¹H NMR 300 MHz (DMSO-d₆) 11.38 (1H, s), 9.81 (1H, s), 7.91(1H, s), 7.70 (1H, s), 7.56 (1H, d, J=7.56 Hz), 7.44 (1H, d, J=8.07 Hz),7.12 (1H, td, J=1.09, 7.59 Hz), 7.08 (1H, s), 7.01 (1H, td, J=0.95, 7.43Hz); ¹³C NMR 300 MHz (DMSO-d₆): 150.05, 136.53, 132.68, 132.48, 129.24,128.05, 124.78, 121.98, 120.27, 119.28, 114.24, 111.97, 111.37, 103.44;(cESI-MS) m/z [M]⁺ calcd for C₁₄H₁₀Br₂NO⁺ 367.91, found 368.1; m.p.104-110° C.

4. Benzimidazole Series (FIGS. 48-49)

2-(1H-benzo[d]imidazole-2-yl)-5-(4-chlorophenyl)-1,3,4-oxadiazole(HGN-421)

To a 100 mL round bottom flask was added 4-chlorobenzoic acid (470.4 mg,3.0 mmol) and 10 mL thionyl chloride. The solution was heated at refluxfor 2 hours with stirring. Excess thionyl chloride was removed underreduced pressure to yield 4-chlorobenzoyl chloride (484.4 mg). The4-chlorobenzoyl chloride was dissolved in 20 mL pyridine and1H-benzo[d]imidazole-2-carbohydrazide (287.4 mg, 1.63 mmol) was added.The resulting solution was stirred and heated at reflux for 6 hours. Thesolution was cooled to room temperature and excess pyridine was removedunder reduced pressure to yield crudeN′-(4-chlorobenzoyl)-1H-benzo[d]imidazole-2-carbohydrazide.N′-(4-chlorobenzoyl)-1H-benzo[d]imidazole-2-carbohydrazide was scrapedoff the flask walls to the bottom of the round bottom flask andpolyphosphoric acid was added to completely cover the product. Themixture was heated to 150° C. until the polyphosphoric acid was lessviscous, and a stirbar was added. The mixture was stirred at 150° C. for2 hours. The reaction mixture was poured over crushed ice and allowed tosit until precipitate formed. The solution was neutralized with sodiumbicarbonate and the solid was filtered and collected. Purification wasdone via recrystallization to yield2-(1H-benzo[d]imidazole-2-yl)-5-(4-chlorophenyl)-1,3,4-oxadiazole (121.5mg, 25.1% yield). ¹H NMR 300 MHz (DMSO-d₆) 8.15 (2H, d, J=8.4 Hz),7.77-7.69 (4H, m), 7.38-7.34 (2H, m); ¹³C NMR 300 MHz (DMSO-d₆): 164.22,163.90, 158.40, 137.64, 137.50, 137.33, 130.23, 130.07, 129.14, 129.01,124.40, 122.60, 122.34; m.p. 250-255° C.

(mass spec data not available due to low solubility in hplc grademethanol and acetonitrile)

2-(1H-benzo[d]imidazol-2-yl)-5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazole(HGN-422)

To a 100 mL round bottom flask was added 4-(trifluoromethyl)benzoic acid(570.4 mg, 3.0 mmol) and 10 mL thionyl chloride. The solution was heatedat reflux for 2 hours with stirring. Excess thionyl chloride was removedunder reduced pressure to yield 4-(trifluromethyl)benzoyl chloride(620.7 mg). The 4-(trifluoromethyl)benzoyl chloride was dissolved in 20mL pyridine and 1H-benzo[d]imidazole-2-carbohydrazide (300.2 mg, 1.7mmol) was added. The resulting solution was stirred and heated at refluxfor 6 hours. The solution was cooled to room temperature and excesspyridine was removed under reduced pressure to yield crudeN′-(4-(trifluoromethyl)benzoyl)-1H-benzo[d]imidazole-2-carbohydrazide.N′-(4-(trifluoromethyl)benzoyl)-1H-benzo[d]imidazole-2-carbohydrazidewas scraped off the flask walls to the bottom of the round bottom flaskand polyphosphoric acid was added to completely cover the product. Themixture was heated to 150° C. until the polyphosphoric acid was lessviscous, and a stirbar was added. The mixture was stirred at 150° C. for2 hours. The reaction mixture was poured over crushed ice and allowed tosit until precipitate formed. The solution was neutralized with sodiumbicarbonate and the solid was filtered and collected. Purification wasdone via recrystallization in ethanol to yield2-(1H-benzo[d]imidazol-2-yl)-5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazole(203.6 mg, 36% yield). ¹H NMR 300 MHz (DMSO-d₆) 8.38-8.33 (2H, m),8.11-8.00 (2H, m), 7.74-7.70 (2H, m), 7.36-7.32 (2H, m); ¹³C NMR 300 MHz(DMSO-d₆): 164.01, 163.82, 159.06, 139.48, 137.93, 132.48, 132.05, (C—Fq 128.17, 126.00, 124.15, 122.39, J=144.5 Hz) 127.43, 127.31, 127.02,126.97, 126.91, 116.88; m.p. 275-280° C.

LIST OF ABBREVIATIONS

¹H-NMR Proton Nuclear Magnetic Resonance

¹³C-NMR Carbon-13 Nuclear Magnetic Resonance

AtIspD Arabidopsis thaliana IspD

AtIspF Arabidopsis thaliana IspF

ATP Adenosine triphosphate

BC Bacillus cereus

BpIspF Burkholderia pseudomallei

BT Burkholderia thailandensis

CDP-ME 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol

CDP-MEP 2-phospho-4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol

cESI-MS Capillary Electrospray Ionization Mass Spectrometry

CP Corynebacterium pseudodiphtheriae

CTP Cytidine triphosphate

CX Corynebacterium xerosis

DMAPP Dimethylallyl diphosphate

DMSO-d₆ Deuterated Dimethyl Sulfoxide

DSF Differential Scanning Fluorimetry

DXS 1-deoxy-D-xylulose-5-phosphate synthase

EC Escherichia coli

FOL7082 Fragments of Life 7082

GHMP galactose/homoserine/mevalonate/phophomevalonate

HMG 3-hydroxy-3-methylglutaryl

IC₅₀ Half Maximal Inhibitory Concentration

IPP Isopentenyl diphosphate

KP Klebsiella pneumonia

LeIspE Lycopersicon esculentum IspE

MaIspE Mycobacterium abscessus IspE

MEcPP 2-C-methyl-D-erythritol 2,4-cyclodiphosphate

MEP 2-C-methyl-D-erythritol 4-phosphate

ML Micrococcus luteus

MPD MVA diphosphate decarboxylase

MpIspD Mycobacterium paratuberculosis IspD

MS Mycobacterium smegmatis

MtIspD Mycobacterium tuberculosis IspD

MVA Mevalonic acid

NADPH Nicotinamide adenine dinucleotide phosphate

NOE Nuclear overhauser effect

PA Pseudomonas aeruginosa

PfIspD Plasmodium falciparum IspD

PMK phospho-MVA kinase

QSAR Quantitative Structure Activity Relationship

SAR Structure Activity Relationship

STD Saturation Transfer Difference

ADMET Absorption, distribution, metabolism, and excretion toxicity

Acetyl-CoA Acetyl-CoA

Amp Ampicillin

BSAT Biological select agents and toxins

CD Circular dichroism

CDC Center for Disease Control and Prevention

CDP Cytidine diphosphate

CMP Cytidine monophosphate

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DOXP 1-deoxy-D-xylulose 5-phosphate

DTT Dithiothreitol

FDA US Food and Drug Administration

FPLC Fast protein liquid chromatography

FSAP Federal Select Agent Program

ΔH°_(obs) Change in enthalpy

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HGN Hagen Compound Code

(His)₆ Hexahistidine peptide affinity tag

HMBDP (E)-4-hydroxy-3-methylbut-2-enyl diphosphate

HMG-CoA 3-hydroxy-3-methyl-glutaryl-coenzyme A

HPLC High performance liquid chromatography

IMAC Immobilized metal affinity chromatography

IPTG Isopropyl β-D-1 thiogalactopyranoside

IspC 1-deoxy-D-xylulose 5-phosphate reductoisomerase

IspD 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase

IspE 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase

IspF 2-C-methyl-D-erythritol 2,4-cylcodiphosphate synthase

IspG 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase

IspH 4-hydroxy-3-methylbut-2-enyl diphosphate reductase

ITC Isothermal titration calorimetry

Kan Kanamycin

K_(B) Binding affinity

K_(D) Dissociation constant

LB Lysogeny broth

MEcDP 2C-methyl-D-erythritol 2,4-cyclodiphosphate

MESG 2-amino-6-mercapto-7-methylpurine riboside

MgCl₂ Magnesium Chloride

mRNA Messenger ribonucleic acid

n Stoichiometry

NaN₃ Sodium azide

NaCl Sodium chloride

OD₆₀₀ Optical density at 600 nm

PABA Para-aminobenzoic acid

PBS Phosphate buffered saline

PCR Polymerase chain reaction

PDB Protein databank

PNP Purine nucleoside phosphorylase

RNA Ribonucleic acid

T_(m) Melting temperature

TEV Tobacco etch virus

TRIS Trishydroxyaminomethane

tRNA Transfer ribonucleic acid

USDA US Department of Agriculture

UV Ultra-violet

Methods

Expression of Escherichia coli (Ec) and Burkholderia thailandensis (Bt)IspD

The gene of EclspD, in the parent vector pET-14b with the restrictionenzymes BgI II at the 5′ and the NcoI at the 3′ and a hexahistidinepeptide tobacco etch virus affinity ((His)₆-TEV) tag at the N′-terminal(obtained from Odom lab) was transformed into BL21(DE3) competent cells.The gene of Bt IspD was obtained from the SSGCID (ButhA.00168.a) withinthe AVA0421 vector (a pET-14b derivative), which possesses ampicillinresistance and a N′-terminal (His)₆-3C tag. The sequences of the vectorsand genes were verified at University of Chicago CRC-DNA sequencingfacility. Once transformed with the respective plasmids, the cells aregrown on a Lysogeny broth (LB) agar plate with 100 μg/mL ampicillin(amp). A single colony was selected and added to 5 mL of LB with 100μg/mL amp and grown overnight at 37° C. with shaking (235 rpm). A 50 mLsubculture was inoculated using the 5 mL overnight culture, which wassubsequently used to inoculate a 1 L LB/amp culture. Cells were grown at37° C. at 235 RPM until the optical density at 600 nm (OD₆₀₀),determined with a Novaspec® II visible spectrophotometer (PharmaciaBiotech), reached a mid-log phase reading 0.5-0.8. Once at mid-logphase, the cells were induced with 1 mM IPTG for 16 hours at 8° C. at235 RPM.⁷⁶ The cells were pelleted via centrifugation at 8,000 RPM for15 minutes and stored at −20° C.

Purification of Escherichia coli (Ec) and Burkholderia thailandensis(Bt) IspD

The frozen cells were resuspended in 10 mM Trishydroxyaminomethane(TRIS) buffer and lysed by sonication using a Digital Sonifier 450®(Branson) for six cycles of 20 seconds on/60 seconds off. Oncesonicated, the sample was centrifuged at 15,000 RPM for 20 minutes at 4°C. to separate the cell remains and insoluble proteins from the solublefraction containing IspD. The supernatant was loaded onto a HisTrap HPnickel affinity IMAC column (GE Healthcare Life Sciences) using aBioLogic LP (Bio-Rad Life Sciences) fast protein liquid chromatography(FPLC) systems. The loading buffer was 20 mM imidazole, pH 8.0, 50 mMsodium phosphate, 500 mM NaCl, and 1 mM dithiothreitol (DTT). Theprotein was eluted using a linear gradient of elution buffer (500 mMimidazole, pH 8.0, 50 mM sodium phosphate, 500 mM NaCl, and 1 mMdithiothreitol (DTT). The fractions containing the IspD enzyme werecombined and purified further with a Superdex 75 HiLoad 26/60 sizeexclusion column (GE Healthcare Life Sciences) using 20 mM TRIS, 150 mMNaCl and 1 mM DTT.

Thermal Stability Determined Using Circular Dichroism

Protein secondary structure can be observed using far ultra-violet (UV)circular dichroism (CD). This method measures the difference between theright and left-handed circularly polarized light in the far UV range(190-250 nm). By following the observed loss of signal related tosecondary structure as the temperature was increased, the percent ofprotein in the native or unfolded form was determined.

All protein samples were dialyzed overnight in 4 L of 20 mM sodiumphosphate with 150 mM NaCl at pH 7.0. CD experiments were performedusing a Circular Dichroism Spectrophotometer 215 (AVIV Biomedical) witha quartz cuvette containing a 1 mm path length. Wavelength scans weredetermined from 200 to 250 nm at 25° C., both before and after thermalunfolding. Thermal unfolding scans were read at either 208 nm for EcIspD or 218 nm for Bt IspD over the temperature range of 25 to 100° C.at 1° C. increments and a temperature equilibrium time of one minute,with a bandwidth of one nanometer and an averaging time of one second.OriginPro 8 Boltzmann fit was used to determine the melting temperature(T_(m)) of each protein.

Thermal Stability Determined Using Differential Scanning Fluorimetry

While CD is a useful technique for observing thermal unfolding ofproteins, there are limitations. One of the greatest limitations is thatCD typically allows only one sample to be run at a time, and each samplecan take several hours to run. Differential scanning fluorimetry (DSF)can follow thermal unfolding of multiple protein samples, at lowervolumes, and at a much faster rate than CD. Similar to a CD observedprotein thermal melt, DSF will determine the fraction of protein innative and unfolded forms. However, while CD directly measures theprotein itself, DSF measures fluorescence of a reporter dye. As aprotein unfolds, hydrophobic residues are exposed that interact with theadded dye, causing a change in the dye molecule's fluorescence emissionprofile. The point of 50% native and 50% unfolded is the considered themelting temperature of the protein.

Ligand-protein binding events can be evaluated using DSF by observingthe change in melting temperature in the presence and absence of ligand.If a ligands presence increases the melting temperature, the ligand isconsidered to be a stabilizing compounds, which most often suggestsbinding to the native form. A decrease in melting temperature wouldindicate a destabilization of the protein. While the observation ofthermal stabilization indicates binding, it does not reveal where thecompound binds, whether it binds to the active site, an allosteric site,or even a crevice on the protein surface unrelated to enzyme activity;each would result in stabilization of the protein's native state.

All DSF experiments were performed in 50 mM phosphate buffer at pH 7.4,150 mM NaCl, 1×SYPRO orange, 2% DMSO and 200 to 1000 nM of the enzymewith a total volume of 25 μL. Final enzyme concentrations used were 2.0μM for Ec IspD and 0.5 μM for Bt IspD. Samples were measured on aBio-Rad iQ5 Real-Time PCR (Bio-Rad Life Sciences) using a temperaturerange of 25 to 95° C. at a rate of 1° C. per minute with thefluorescence measured every 0.5° C. every 10 seconds with wavelengths of530 and 570 nm used for excitation and emission, respectively. Themelting temperature (Tm value) was determined from the first derivativeof the graph of relative fluorescence units vs. temperature.

Binding Studies with Isothermal Titration Calorimetry

Binding studies are essential for determining what type of affinity aprotein has for a ligand. Isothermal titration calorimetry (ITC) allowsthe ability to determine the binding affinity (K_(B)) which ischaracterized as the ratio of the protein (or macromolecule) ligandcomplex concentration [ML] by the concentration of free macromolecule[M] and ligand [L], as presented in the following equation:

$\begin{matrix}{K_{B} = {\frac{\lbrack{ML}\rbrack}{\lbrack M\rbrack\lbrack L\rbrack}.}} & {{{Eq}.\mspace{14mu} 2}\text{-}1}\end{matrix}$

The equilibrium dissociation constant (K_(D)) can be obtained by takingthe inverse of the binding affinity constant:

$\begin{matrix}{K_{D} = {\frac{1}{K_{B}}.}} & {{{Eq}.\mspace{14mu} 2}\text{-}2}\end{matrix}$

Along with the binding affinity, the enthalpy change (ΔH°_(obs)) andstoichiometry (n) are determined directly from an ITC experiment, whichalso allows calculation of the change in entropy (ΔS°_(obs)).

ITC studies were performed with a Microcal VP-ITC titration calorimeter(Malvern Instruments). Proteins were dialyzed overnight against fourliters of buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4).Compounds were dissolved in DMSO and brought to concentration with thedialysis buffer, with a maximum DMSO concentration of 10% by volume, andcentrifuged at 10,000 RPM for 10 minutes. DMSO concentrations werematched between the protein and ligand. The dialysis buffer was used forall sample dilutions to ensure buffers matching between the titrant andtitrate, thus minimizing the excess heat due to buffer mismatch. ThePace method was utilized for the determination of the extinctioncoefficients at 280 nm for Ec IspD (30,940 M⁻¹ cm⁻¹) and Bt IspD (41,940M⁻¹ cm⁻¹). Cytidine triphosphate's extinction coefficient was 9,100 M⁻¹cm⁻¹. The absorbance values of protein and CTP solutions were determinedusing the NanoDrop (in triplicate). All other compound concentrationswere determined by dry weight. ITC experiments were performed with IspDin the cell and ligand in the syringe, which is at 100 times theconcentration of the sample in the cell due to the low predictedaffinities for IspD.

The initial injection volume was 2 μL (omitted) followed by 27 10 μLinjections, every 240 seconds. Samples were run at 25° C., at pH 7.4,with a stirring speed of 307 RPM. Dilution runs were performed to allowdetermination of background heats caused by solution mismatch andinstrument noise. Dilution runs were performed with ligand (at the sameconcentration as in the experiment run) in the syringe titrated into thedialysis buffer in the cell. Data were fit using OriginPro 7 with theITC add-on by the manufacturer.

Enzyme Inhibition Determined Using EnzChek® Phosphate Assay Kit

Thermal shift assays allow observation of binding. However, binding doesnot always equate to activity. An enzymatic assay typically follows adecrease in reactant concentration, an increase in productconcentration, or the formation of a byproduct. Here, for theplate-based assay, IspD converts MEP to CDP-ME pyrophosphate, which inturn is converted to inorganic phosphate (P_(i)) with the enzymeinorganic pyrophosphatase. The EnzChek® Phosphate Assay kit follows theformation of the inorganic phosphate through a reaction with2-amino-6-mercapto-7-methylpurine riboside (MESG) and the enzyme purinenucleoside phosphorylase (PNP). This reaction converts the MESG intoribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine resulting in aspectrophotometric shift in a maximum absorbance from 330 nm to 360 nm.Reactions were performed in 50 μL final volumes in 96-well clear flatbottom plates, and were initiated by addition of 50 ng purified Ec IspD.Absorbance at 360 nm was measured every 31 seconds for 20 minutes on aSynergy 2 Multi-Mode plate reader (BioTek), preheated to 37° C. Finalconcentration of reagents was as follows: 1×EnzChek® reaction buffer (50mM Tris pH 7.5, 1 mM magnesium chloride (MgCl₂), 100 μM sodium azide(NaN₃)), 0.2 mM MESG, 1 U/ml PNP, 1 U/mL inorganic Pyrophosphatase(NEB), 200 mM CMP (Sigma), and 50 μM MEP (Echelon).

Purification of Ec and Bt IspD

Both Escherichia coli and Burkholderia thailandensis IspD were purifiedusing a nickel affinity column with a 20-500 mM gradient elution usingimidazole. Fractions associated with the UV absorbance peak in thechromatogram, verified with a NanoDrop 2000c spectrophotometer (ThermoScientific), were combined and concentrated to 10 mL using a spinconcentrator. The resulting concentrate was further purified using asize exclusion chromatography column (Superdex 75 HiLoad 26/60), andfractions related to the UV absorbance peak of IspD were collected,combined, and concentrated. The protein was aliquoted and stored at −80°C. until needed for thermal shift (DSF), circular dichroism andenzymatic assays.

Melting Point Determination of IspD

Circular Dichroism was used to determine the thermal stability of Ec andBt IspD. Prior to performing CD, 20 mM of each protein was dialyzed infour liters of buffer (20 mM phosphate at pH 7.0 and 150 mM NaCl)overnight. Wavelength scans were run before the thermal melt to get abaseline reading which confirmed the mainly alpha helical secondarystructure of the proteins. Full scans were repeated post-thermal melt tocharacterize the protein's reversibility. Both species show loss ofsecondary structure which may suggest the thermal unfolding to benon-reversible under the conditions of the experiment. The thermalstability of each of the proteins was observed at a single wavelengthover a temperature range of 25−100° C. The melting temperature (T_(m))was determined to be approximately 66° C. for Ec IspD and 53.5° C. forBt IspD, which will be used as reference temperatures for furtherthermal shift assays.

Thermal Shift of IspD to Determine Binding of Compounds

Prior to testing compounds directly against IspD, a concentration testwas performed to determine the lowest concentration of IspD that wouldstill provide a reasonable change in signal upon unfolding. Comparingthe melting temperature with and without the presence of compound canresult in a thermal shift, which may indicate binding. The oxadiazoleseries were evaluated with very little observed thermal shifts againstEc IspD.

Binding Studies of Bt IspD with CTP

ITC studies were performed to observe the dissociation constant alongwith thermodynamic properties of the reactant CTP with Bt IspD. Due tothe low affinity, the binding stoichiometry was fixed at two CTPs perdimer during data analysis. The ΔH° of CTP showed to have a value of−6.991±1.749 kcal/mol and an unfavorable entropic contribution of −TΔSof 5.6±1.838. K_(D app) of CTP was shown to be in the millimolar range.

TABLE 1 Thermal Shift Assay Results -Compounds tested againstMycobacterium tuberculosis IspD (MtIspD) and Lycopericon esculentum IspE(LeIspE). MtIspD LeIspE melting melting MtIspD point LeIspE pointmelting change melting change Sample Structure temp (° C.) (° C.) temp(° C.) (° C.) HGN-207

49.5 1.5 48.25 −1.25 HGN-209

49.5 1.5 47.25 −2.25 HGN-250

48.8 0.8 46.75 −2.75 HGN-240

48.8 0.8 48.00 −1.50 HGN-239

49.5 1.5 47.00 −2.50 HGN-211

49.3 1.3 48.75 −0.75 HGN-252

49.5 1.5 47.00 −2.50 HGN-254

48.5 0.5 47.50 −2.00 HGN-241

48.5 0.5 47.75 −1.75 HGN-213

48.8 0.8 47.50 −2.00 HGN-214

49.8 1.8 48.50 −1.00 HGN-242

48.8 0.8 47.75 −1.75 HGN-223

48.0 0.0 49.25 −0.25 HGN-339

49.3 1.3 47.50 −2.00 HGN-337

48.8 0.8 47.75 −1.75 HGN-338

48.0 0.0 48.00 −1.50 HGN-336

49.5 1.5 48.25 −1.25 HGN-333

49.3 1.3 47.75 −1.75 HGN-334

48.0 0.0 48.00 −1.50 HGN-335

48.5 0.5 48.00 −1.50 HGN-421

48.8 0.8 48.00 −1.50 HGN-422

49.0 1.0 46.25 −3.25 HGN-221

48.5 0.5 49.50 0.00 HGN-215

48.8 0.8 47.50 −2.00 HGN-287

48.5 0.5 49.25 −0.25 HGN-288

49.0 1.0 48.00 −1.50 HGN-340

49.3 1.3 50.00 0.50

TABLE 2 Lettuce Leaf Assay Results- In vivo assay results of compoundstested against lettuce seeds to test for bleaching, root developmentinhibition, and germination inhibition. Ranking is a qualitative measurefrom 0 (no effect) to 5(complete inhibition or bleaching). Root SampleStructure Conc. Bleaching Dev. Germination HGN-207

 1.0 mM  0.5 mM 3 2 0 0 2 2 HGN-209

 1.0 mM  0.5 mM 0 0 1 1 2 1 HGN-250

 1.0 mM  0.5 mM 0 0 0 0 3 1 HGN-240

 1.0 mM  0.5 mM 0 0 0 0 2 1 HGN-239

 1.0 mM  0.5 mM 0 0 0 0 1 0 HGN-211

 1.0 mM  0.5 mM 0 0 0 0 2 1 HGN-252

 1.0 mM  0.5 mM 0 0 0 0 2 1 HGN-254

 0.3 mM 0.15 mM 0 0 0 0 0 0 HGN-241

 0.2 mM 0 0 1 HGN-213

 0.2 mM 0 0 1 HGN-214

 1.0 mM  0.5 mM  0.2 mM 0 0 0 0 0 0 3 1 0 HGN-242

 1.0 mM  0.5 mM 0 0 0 0 4 2 HGN-223

 1.0 mM  0.5 mM 0 0 0 0 4 1 HGN-339

 0.3 mM 0.15 mM 0 0 0 0 0 0 HGN-337

 0.3 mM 0.15 mM 0 0 0 0 0 0 HGN-338

 0.3 mM 0.15 mM 0 0 0 0 1 0 HGN-336

 0.3 mM 0.15 mM 0 0 0 0 0 1 HGN-333

 0.3 mM 0.15 mM 0 0 0 0 0 0 HGN-334

 0.3 mM 0.15 mM 0 0 0 0 0 0 HGN-335

 0.2 mM  0.1 mM 0 0 0 0 0 0 HGN-421

 0.3 mM 0.15 mM 0 0 0 0 1 1 HGN-422

 0.3 mM 0.15 mM 0 0 0 0 0 0 HGN-221

 0.3 mM 0.15 mM 0 0 0 0 0 0 HGN-215

 0.3 mM 0.15 mM 0 0 0 0 0 0 HGN-287

 0.3 mM 0.15 mM 0 0 0 0 0 0 HGN-288

 0.2 mM  0.1 mM 0 0 0 0 0 0 HGN-340

 0.3 mM 0.15 mM 0 0 0 0 0 0

TABLE 3 Assay Results - Compounds were tested against AtIspD, LeIspE,and AtIspF and results are given as IC50 values. (University of Hamburg,Boris Illarionov, director Markus Fischer)

AtIspD LeIspE AtIspF HGN # X Y R₁ IC₅₀, μM IC₅₀, μM IC₅₀, μM 207 C NHR—H >1000 >1000 >1000 209 C NH R—CH₃ >1000 >1000 >1000 250 C NH

>1000 >1000 >1000 240 C NH

>1000 >1000 >1000 239 C NH

>1000 >1000 >1000 212 C NH

>1000 >1000 >1000 211 C NH R—CF₃ >1000 >1000 >1000 251 C NH

>1000 >1000 >1000 252 C NH

>1000 >1000 >1000 254 C NH

322 ± 14 >1000 >1000 208 C NH

342 ± 42 165 ± 47 >1000 241 C NH

>1000 149 ± 41 >1000 213 C NH

490 ± 91 326 ± 29 >1000 214 C NH

>1000 269 ± 23 >1000 242 C NH

225 ± 17 109 ± 35 >1000 223 C NH

209 ± 29 126 ± 12 >1000 339 N S

158 ± 14 >500 >500 337 N S

>500 >500 >500 338 N S

>500 >500 >500 336 N S

>500 493 ± 49 >500 333 N S

151 ± 28  31 ± 17 291 ± 26 334 N S

>500 >500 >500 335 N S

>500 >500 >500 421 N NH

 88 ± 20 298 ± 49 484 ± 95 422 N NH

251 ± 21 267 ± 12 >500 221 C NH

636 ± 68 >1000 >1000 215 C NH

>1000 430 ± 48 >1000 287 C NH

501 ± 82 >1000 >1000 288 C NH

255 ± 35 >1000 >1000

HGN # AtIspD IC₅₀, μM LeIspE IC₅₀, μM AtIspF IC₅₀, μM 340 50 ± 2 >500470 ± 116

TABLE 4 Results from Antibacterial Preliminary Screening- Compounds aregiven scores from 0-3 in the preliminary screening in which 0 representsno inhibition and 3 is comparable to commercial antibiotic control. HGN#Structure BT MS CX CP EC KP ML BC PA 207

0 0 0 0 0 0 0 0 0 209

0 0 0 0 0 0 0 0 0 250

0 0 0 0 0 0 0 0 0 240

0 0 0 0 0 0 0 0 0 239

0 0 0 0 0 0 0 0 0 212

0 0 0 0 0 0 0 0 0 211

0 0 0 0 0 0 0 0 0 251

0 0 0 0 0 0 0 0 0 252

0 0 0 0 0 0 0 0 0 254

0 0 0 0 0 0 0 0 0 208

0 0 0 0 0 0 0 0 0 241

0 0 0 0 0 0 0 0 0 213

0 0 0 0 0 0 0 0 0 214

0 0 0 0 0 0 0 0 0 242

0 0 0 0 0 0 0 0 0 223

0 0 0 0 0 0 0 0 0 339

0 0 0 0 0 0 0 0 0 337

0 0 0 0 0 0 0 0 0 338

0 0 0 0 0 0 0 0 0 336

0 0 0 0 0 0 0 0 0 333

0 0 0 0 0 0 0 0 2 334

0 0 0 0 0 0 0 0 0 335

0 0 0 0 0 0 0 0 1 421

1 1 0 0 0 0 0 0 0 422

1 1 0 0 0 0 0 0 0 221

0 0 0 0 0 0 0 0 0 215

0 0 0 0 0 0 0 0 0 287

0 0 0 0 0 0 0 0 0 288

0 0 0 0 0 0 0 0 0 340

0 0 0 0 0 0 0 2 0 BT = Burkholderia thailandensis, MS = Mycobacteriumsmegmatis, CX = Corynebacterium xerosis, CP = Corynebacteriumpseudodiphtheriae, EC = Escherichia coli, KP = Klebsiella pneumonia, ML= Micrococcus luteus, BC = Bacillus cereus, PA = Pseudomonas aeruginosa.(University of Northern Illinois, Debarati Gose and R. Meganathan)

Antibacterial assay results for additional oxadiazole compounds shown inTable 5. The data obtained from this assay was determined by theorganisms' growth and zone of inhibition, measured in millimeters, whenintroduced to a compound. The antibacterial assays show the zone ofinhibition of each of the nine organisms.

TABLE 5 Results from Antibacterial Preliminary Screening- Compounds aregiven scores from 0-3 in the preliminary screening in which 0 representsno inhibition and 3 is comparable to commercial antibiotic control.

HGN Conc. Antibacterial # X Y Z R (mM) Bt Pa Ml Kp Ec Bc Ms Cx Cp 811 CNH O

0.1 0.5 1    0  8 12 0 0 0 0 0 0 0 0 0 0 0 0  0   12.5 16 0 0 0 0 0 0 00 0 810 C NH O

0.1 0.5 1    0   11.5 17 0 0 0 0 0 0 0 0 0 0 0 0  0 11   16.5 0 0 0 0 00 0 0 0 824 N S CH₂

0.1 0.5 1    0  0  0 0 0 0 0 0 0 0 0 0 0 0 0  0  0  0 0 12  18  0 0 0 00 10  840 N S O

0.1 0.5 1    0 14   17.5 0 0 0 0 0 0 0 0  16.5 0 0 0  0  0   14.5 0 0 00 0 0 0 12  19  826 N S O

0.1 0.5 1    0  0  0 0 0 0 0 0 0 0 0 0 0 0 0  0  0 12 0 13  20  0 0 0 00 0 825 N S O

0.1 0.5 1    0  0  0 0 0 0 0 0 11  0 0 0 0 0 0  0  0   11.5 0 13  18  00 0 0 0 0 833 N S O

0.1 0.5 1    0 12 17 0 0 0 0 0 13  0 0 0 0 0 0  0 10 15 0 0 0 0 0 15  00 14  834 N S O

0.1 0.5 1    0  0  0 0 0 0 0 0 0 0 0 0 0 0 0  0 11 18 0 0 0 0 0 0 0 0 0835 N S O

0.1 0.5 1    0   12.5 18 0 0 0 0 0 14  0 14  18  0 0 0  0 14 20 0 0 0 00 0 0 0 0 836 N S O

0.1 0.5 1    0 15 18 0 0 0 0 0 0 0 0 7 0 0 NT  0  0 13 0 0 0 0 0 11  0 00 333 N S O

0.1 0.5 1    0 15 19 0 18  25  0 0 0 0 0 0 0 0 0  0  0   12.5 0 0 0 0 00 0 0 0 837 N S O

0.1 0.5 1    0 13   19.5 0 0 0 0 6 10  0 0 0 0 10  17   0 13 19 0 0 0 011  17  0 0 0 Bt = Burkholderia thailandensis, Ms = Mycobacteriumsmegmatis, Cx = Corynebacterium xerosis, Cp = Corynebacteriumpseudodiphtheriae, Ec = Escherichia coli, Kp = Klebsiella pneumonia, Ml= Micrococcus luteus, Bc = Bacillus cereus, Pa = Pseudomonas aeruginosa.

TABLE 6 Primary Screen Target Organism SSGCID ID Hits IspD Mycobacteriumparatuberculosis MypaA.00168.a 102 IspE Mycobacterium abscessusMyabA.00725.a 176 IspF Burkholderia pseudomallei BupsA.00122.a 72control Toxoplasma gondii TogoA.00914.a 81

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We claim:
 1. A method to inhibit bacteria, the method comprisingcontacting the bacteria with a compound of the formula I

wherein X is NH or S; Y is CH or N; Z is selected from the groupconsisting of C₁-C₆ alkylene, C₂-C₆ alkenylene, and C₃-C₆ cycloalkylene,or Z is absent; and R is selected from the group consisting of halogen,—OH, —NO₂, C₂-C₆ alkenyl, —O(C₁-C₆ alkyl), and —O(C₆-C₁₀ aryl), whereineach hydrogen atom in C₂-C₆ alkenyl, and —O(C₁-C₆ alkyl) isindependently optionally substituted by halogen, —OH, or —NO₂, andwherein each hydrogen atom in —O(C₆-C₁₀ aryl) is optionally substitutedby —OH.